RESUMEN
Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials.
Asunto(s)
Dependovirus/genética , Terapia Genética/efectos adversos , Vectores Genéticos/genética , Leucodistrofia de Células Globoides/complicaciones , Leucodistrofia de Células Globoides/terapia , Animales , Trasplante de Médula Ósea/métodos , Carcinoma Hepatocelular/etiología , Terapia Combinada , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Neoplasias Hepáticas/etiología , RatonesRESUMEN
Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing "psychosine hypothesis." We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients. We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized l-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease.
Asunto(s)
Ceramidasa Ácida/genética , Eliminación de Gen , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Psicosina/metabolismo , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Metilación de ADN , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Leucodistrofia de Células Globoides/tratamiento farmacológicoRESUMEN
Globoid-cell leukodystrophy (GLD) is an inherited demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC). A previous study in the murine model of GLD (twitcher) demonstrated a dramatic synergy between CNS-directed adeno-associated virus 2/5 (AAV2/5) gene therapy and myeloreductive bone marrow transplantation (BMT). However, the mechanism by which these two disparate therapeutic approaches synergize is not clear. In addition, the therapeutic efficacy may have been limited since the CNS-directed gene therapy was restricted to the forebrain and thalamus. In the current study, intrathecal and intracerebellar injections were added to the therapeutic regimen and the mechanism of synergy between BMT and gene therapy was determined. Although AAV2/5 alone provided supraphysiological levels of GALC activity and reduced psychosine levels in both the brain and spinal cord, it significantly increased CNS inflammation. Bone marrow transplantation alone provided essentially no GALC activity to the CNS and did not reduce psychosine levels. When AAV2/5 is combined with BMT, there are sustained improvements in motor function and the median life span is increased to 123 d (range, 92-282 d) compared with 41 d in the untreated twitcher mice. Interestingly, addition of BMT virtually eliminates both the disease and AAV2/5-associated inflammatory response. These data suggest that the efficacy of AAV2/5-mediated gene therapy is limited by the associated inflammatory response and BMT synergizes with AAV2/5 by modulating inflammation.
Asunto(s)
Trasplante de Médula Ósea/métodos , Encéfalo/metabolismo , Terapia Genética/métodos , Inflamación/terapia , Leucodistrofia de Células Globoides/terapia , Médula Espinal/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Dependovirus/genética , Imagen de Difusión Tensora/métodos , Modelos Animales de Enfermedad , Citometría de Flujo/métodos , Galactosilceramidasa/biosíntesis , Galactosilceramidasa/deficiencia , Vectores Genéticos/fisiología , Indoles , Inflamación/etiología , Estimación de Kaplan-Meier , Leucodistrofia de Células Globoides/complicaciones , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patología , Longevidad/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido Peryódico , Psicosina/metabolismo , Temblor/etiologíaRESUMEN
Enzyme replacement therapy (ERT) with intravenous recombinant human alpha-l-iduronidase (IV rhIDU) is a treatment for patients with mucopolysaccharidosis I (MPS I). Spinal cord compression develops in MPS I patients due in part to dural and leptomeningeal thickening from accumulated glycosaminoglycans (GAG). We tested long-term and every 3-month intrathecal (IT) and weekly IV rhIDU in MPS I dogs age 12-15months (Adult) and MPS I pups age 2-23days (Early) to determine whether spinal cord compression could be reversed, stabilized, or prevented. Five treatment groups of MPS I dogs were evaluated (n=4 per group): IT+IV Adult, IV Adult, IT + IV Early, 0.58mg/kg IV Early and 1.57mg/kg IV Early. IT + IV rhIDU (Adult and Early) led to very high iduronidase levels in cervical, thoracic, and lumber spinal meninges (3600-29,000% of normal), while IV rhIDU alone (Adult and Early) led to levels that were 8.2-176% of normal. GAG storage was significantly reduced from untreated levels in spinal meninges of IT + IV Early (p<.001), IT+IV Adult (p=.001), 0.58mg/kg IV Early (p=.002) and 1.57mg/kg IV Early (p<.001) treatment groups. Treatment of dogs shortly after birth with IT+IV rhIDU (IT + IV Early) led to normal to near-normal GAG levels in the meninges and histologic absence of storage vacuoles. Lysosomal storage was reduced in spinal anterior horn cells in 1.57mg/kg IV Early and IT + IV Early animals. All dogs in IT + IV Adult and IV Adult groups had compression of their spinal cord at 12-15months of age determined by magnetic resonance imaging and was due to protrusion of spinal disks into the canal. Cord compression developed in 3 of 4 dogs in the 0.58mg/kg IV Early group; 2 of 3 dogs in the IT + IV Early group; and 0 of 4 dogs in the 1.57mg/kg IV Early group by 12-18months of age. IT + IV rhIDU was more effective than IV rhIDU alone for treatment of meningeal storage, and it prevented meningeal GAG accumulation when begun early. High-dose IV rhIDU from birth (1.57mg/kg weekly) appeared to prevent cord compression due to protrusion of spinal disks.
Asunto(s)
Terapia de Reemplazo Enzimático/veterinaria , Iduronidasa/uso terapéutico , Mucopolisacaridosis I/tratamiento farmacológico , Mucopolisacaridosis I/veterinaria , Compresión de la Médula Espinal/tratamiento farmacológico , Compresión de la Médula Espinal/veterinaria , Animales , Perros , Humanos , Inyecciones Espinales , Imagen por Resonancia Magnética/veterinaria , Médula Espinal/patología , Compresión de la Médula Espinal/patologíaRESUMEN
Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme beta-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Terapia Genética/métodos , Lentivirus/genética , Enfermedades por Almacenamiento Lisosomal/genética , Células Madre Mesenquimatosas/citología , Mucopolisacaridosis VII/terapia , Animales , Electrorretinografía/métodos , Glucuronidasa/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mucopolisacaridosis VII/genética , Trasplante HeterólogoRESUMEN
Granular cell tumor is a benign tumor of likely neural or neuroectodermal origin that occurs most commonly in the subcutaneous tissues of the trunk, breast, and extremities of adults. Congenital gingival lesions comprise the majority of the pediatric granular cell tumors. Granular cell tumors are generally small and asymptomatic, and while 1 in 10 patients has multiple tumors, recurrence and malignancy are very rare. Mediastinal granular cell tumors have been reported, most occurring in young adult or middle-aged women. We present a case of a 16-year-old asymptomatic boy with a large mediastinal granular cell tumor incidentally identified after a motor vehicle accident, and we review the intraoperative, microscopic, and ultrastructural features of this tumor. Both the patient's age and anatomical location are unusual for this tumor, which presented technical and diagnostic challenges to the patient care team.
Asunto(s)
Tumor de Células Granulares/patología , Neoplasias del Mediastino/patología , Adolescente , Biomarcadores de Tumor/análisis , Biopsia , Tumor de Células Granulares/química , Tumor de Células Granulares/cirugía , Humanos , Inmunohistoquímica , Hallazgos Incidentales , Imagen por Resonancia Magnética , Masculino , Neoplasias del Mediastino/química , Neoplasias del Mediastino/cirugía , Microscopía Electrónica , Cirugía Torácica Asistida por Video , Resultado del Tratamiento , Carga TumoralRESUMEN
The mucopolysaccharidoses (MPSs) are lysosomal storage diseases resulting from impaired catabolism of sulfated glycosaminoglycans. MPS VII mice lack lysosomal beta-glucuronidase (GUSB) activity, leading to the accumulation of partially degraded chondroitin, dermatan, and heparan sulfates in most tissues. Consequently, these mice develop most of the symptoms exhibited by human MPS VII patients, including progressive visual and cognitive deficits. To investigate the effects of reducing lysosomal storage in nervous tissues, we injected recombinant adeno-associated virus encoding GUSB directly into the vitreous humor of young adult mice. Interestingly, GUSB activity was subsequently detected in the brains of the recipients. At 8-12 weeks after treatment, increased GUSB activity and reduced lysosomal distension were found in regions of the thalamus and tectum that received inputs from the injected eye. Lysosomal storage was also reduced in adjacent nonvisual regions, including the hippocampus, as well as in the visual cortex. The findings suggest that both diffusion and trans-synaptic transfer contribute to the dissemination of enzyme activity within the CNS. Intravitreal injection may thus provide a means of delivering certain therapeutic gene products to specific areas within the CNS.
Asunto(s)
Sistema Nervioso Central/enzimología , Terapia Genética/métodos , Glucuronidasa/genética , Lisosomas/enzimología , Mucopolisacaridosis VII/enzimología , Animales , Sistema Nervioso Central/patología , Sistema Nervioso Central/virología , ADN Viral/análisis , Dependovirus/genética , Modelos Animales de Enfermedad , Vías de Administración de Medicamentos , Activación Enzimática/genética , Ojo/enzimología , Ojo/virología , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Glucuronidasa/biosíntesis , Glucuronidasa/deficiencia , Hipocampo/enzimología , Hipocampo/patología , Hipocampo/virología , Humanos , Lisosomas/patología , Ratones , Ratones Mutantes Neurológicos , Mucopolisacaridosis VII/genética , Mucopolisacaridosis VII/patología , Reacción en Cadena de la Polimerasa , Colículos Superiores/enzimología , Colículos Superiores/patología , Colículos Superiores/virología , Tálamo/enzimología , Tálamo/patología , Tálamo/virología , Resultado del Tratamiento , Corteza Visual/enzimología , Corteza Visual/patología , Corteza Visual/virología , Vías Visuales/enzimología , Vías Visuales/patología , Vías Visuales/virologíaRESUMEN
To block development of progressive childhood diseases, in utero transplantation (IUTx) requires immediate and significant donor peripheral blood (PB) cell amplification. To date, negligible and nontherapeutic donor PB cell levels have been observed postnatally, except in patients with immunodeficiency diseases. Donor cell fate in utero still is not clear. Ease of identifying and quantifying beta-glucuronidase (GUSB)-expressing donor cells in GUSB-null mucopolysaccharidosis type VII (MPSVII) mouse recipients allowed us to evaluate temporal donor cell engraftment and amplification post-IUTx. Like humans, MPSVII mice are unable to catabolize lysosomal glycosaminoglycans and progressively develop severe storage disease unless they are treated early in life.IUTx recipients were nonablated MPSVII fetuses and genetically stem cell-deficient, and hence myeloablated, W(41)/W(41) MPSVII fetuses. Donor GUSB+ cells were identified and counted in histochemical tissue sections. Quantitative results were confirmed by flow cytometry, enzyme analysis, and histopathology. Whereas GUSB+ cells engraft in most tissues in utero, significant amplification does not occur until the first postnatal week in the nonablated MPSVII hosts. In contrast, genetically myeloablated MPSVII recipients display widely distributed donor cell replacement accompanied by extensive amplification in utero. In both models, storage is alleviated in adult tissues with significant donor cell repopulation. To become therapeutic, IUTx must overcome the limitations of donor cell expansion in the highly competitive fetal environment. Fortunately, nonablative mechanisms to amplify cells in utero are coming on line.
Asunto(s)
Enfermedades Fetales/terapia , Glucuronidasa/análisis , Mucopolisacaridosis VII/terapia , Trasplante de Células Madre/métodos , Animales , Recuento de Células , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
Low-grade fibromyxoid sarcoma (previously known as Evans tumor) is a rare soft tissue neoplasm characterized by a deceptively bland appearance despite the potential for late metastasis or recurrence. We describe a 13-year-old patient with a popliteal fossa mass initially thought to be benign that, because of array-comparative genomic hybridization findings and subsequent immunohistochemistry, was diagnosed as low-grade fibromyxoid sarcoma. The array-comparative genomic hybridization demonstrated a loss of 11p11.2p15.5 and a gain of 16p11.2p13.3 with breakpoints involving the CREB3L1 (cAMP responsive element-binding protein 3-like 1) and FUS (fused in sarcoma) genes, respectively. Subsequent fluorescence in situ hybridization analysis of a dual-labeled break-apart FUS probe on interphase cells was positive. Our case highlights the importance of using genetic information obtained via array-comparative genomic hybridization to classify accurately pediatric soft tissue tumors.
Asunto(s)
Hibridación Genómica Comparativa , Fibrosarcoma/genética , Fibrosarcoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Adolescente , Hibridación Genómica Comparativa/métodos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Fibrosarcoma/diagnóstico , Humanos , Masculino , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Globoid cell leukodystrophy (GLD, Krabbe Disease) is a lysosomal storage disease, resulting from the genetic deficiency of galactosylceramidase (GALC). This disease is marked by accumulation of the cytotoxic lipid psychosine (Psy). Psychosine is known to induce oxidative stress in cultured cells, and this stress can be ameliorated through co-treatment with the antioxidant N-acetyl cysteine (NAC). Oxidative stress has also been observed in vivo in the mouse model of GLD, the Twitcher mouse (Twi). We hypothesized that treating oxidative stress with NAC; either alone or in combination with bone marrow transplant (BMT) would improve the course of disease. All breeding cages were maintained on water containing NAC. Once born, the pups received IP boluses of NAC three times per week, and were maintained on NAC-containing water. A separate cohort of animals received the same regimen of NAC in addition to a BMT on post-natal days 2-3. Although NAC lowers the level of oxidized proteins in the brains of Twi mice, and dramatically improves immunohistochemical markers of disease, neither treatment results in any clinical improvements in the Twi mouse. Our data suggest that oxidative stress may be sufficiently down-stream in the pathogenic cascade initiated by Psy accumulation as to be difficult or impossible to treat with standard pharmacologic agents. It is possible that NAC may synergize with other therapies or combinations of therapies. A better understanding of the initiating effects of Psy toxicity and oxidative damage may uncover treatable therapeutic targets.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Trasplante de Médula Ósea , Leucodistrofia de Células Globoides/tratamiento farmacológico , Leucodistrofia de Células Globoides/cirugía , Estrés Oxidativo , Animales , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Estrés Oxidativo/efectos de los fármacosRESUMEN
Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by loss of activity of α-l-iduronidase and attendant accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate. Current treatments are suboptimal and do not address residual disease including corneal clouding, skeletal deformities, valvular heart disease, and cognitive impairment. We treated neonatal dogs with MPS I with intravenous recombinant α-l-iduronidase replacement therapy at the conventional 0.58 mg/kg or a higher 1.57 mg/kg weekly dose for 56 to 81 weeks. In contrast to previous results in animals and patients treated at a later age, the dogs failed to mount an antibody response to enzyme therapy, consistent with the induction of immune tolerance in neonates. The higher dose of enzyme led to complete normalization of lysosomal storage in the liver, spleen, lung, kidney, synovium, and myocardium, as well as in the hard-to-treat mitral valve. Cardiac biochemistry and function were restored, and there were improvements in skeletal disease as shown by clinical and radiographic assessments. Glycosaminoglycan levels in the brain were normalized after intravenous enzyme therapy, in the presence or absence of intrathecal administration of recombinant α-l-iduronidase. Histopathological evidence of glycosaminoglycan storage in the brain was ameliorated with the higher-dose intravenous therapy and was further improved by combining intravenous and intrathecal therapy. These findings argue that neonatal testing and early treatment of patients with MPS I may more effectively treat this disease.
Asunto(s)
Terapia Enzimática , Iduronidasa/administración & dosificación , Iduronidasa/uso terapéutico , Mucopolisacaridosis I/terapia , Animales , Animales Recién Nacidos , Huesos/patología , Encéfalo/metabolismo , Encéfalo/patología , Perros , Glicosaminoglicanos/metabolismo , Humanos , Iduronidasa/genética , Articulaciones/patología , Lisosomas/metabolismo , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/fisiopatología , Distribución TisularRESUMEN
Treatment of nonmalignant childhood disorders by bone marrow transplantation (BMT) is limited by toxicity from preparatory regimens and immune consequences associated with engraftment of allogeneic donor cells. Using costimulatory blockade (anti-CD40L mAb and CTLA-4Ig) combined with high-dose BMT in nonablated neonates, we obtained engraftment and established tolerance using both partially MHC mismatched (H2g7 into H2b) and fully mismatched BM (H2s into H2b). Recipients were mucopolysaccharidosis type VII (MPS VII) mice with lysosomal storage disease in order to assess therapeutic outcome. Recipients treated with donor lymphocyte infusion (DLI) amplified microchimerism to full donor. Recipients without DLI maintained long-term engraftment, tolerance, and had extended life spans. DLI increased donor cell mediated replacement of beta-glucuronidase (GUSB) activity in all tissues and maintained clearance of lysosomes better than in non-DLI-treated mice. DLI amplification of partially mismatched BM and fully mismatched BM caused late onset chronic GvHD in 56% and 100% of recipients, respectively.
Asunto(s)
Trasplante de Médula Ósea , Activación de Linfocitos , Transfusión de Linfocitos , Enfermedades por Almacenamiento Lisosomal/terapia , Agonistas Mieloablativos , Animales , Quimera , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/patología , Cardiopatías/terapia , Inmunohistoquímica , Riñón/patología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Trasplante HomólogoRESUMEN
Lysosomal storage diseases (LSD) respond to bone marrow (BM) transplantation when donor-derived cells deliver needed enzyme. Hypothetically, the ubiquitous resident macrophages (MPhi) are the primary delivery vehicle of therapeutic protein. In mucopolysaccharidosis type VII (MPS VII) mice with LSD, transplanted mature MPhi reduce undegraded glycosaminoglycans (GAG) in the lysosome but are incapable of self-renewal, leading to return of storage after 1 month. We show here that a population of early BM-derived myeloid progenitors devoid of long-term hematopoietic stem cells (LT-HSC) engrafted MPS VII BM, released monocytes into peripheral blood (PBL), and engrafted tissues at known sites of resident MPhi. These primitive Mac-1- cells were sorted from normal whole BM and were defined by ER-MP12hi20-58med/hi labeling. Lysosomal storage was reduced in liver, spleen, thymus, heart, kidney, and bone. Cells persisted for 3 months, suggesting self-renewal capacity or a long half-life. Cells sorted from BM by ER-MP12-20hi marker expression (which are maturer myeloid cells that express Mac-1) engrafted tissues instead of BM and quantitatively repopulated less than cells derived from the ER-MP12hi20-58med/hi population. Also, reduction of lysosomal storage was variable and generally less when compared to that following transplantation of immature ER-MP12hi20-58med/hi cells. We conclude that primitive myeloid progenitors are more therapeutic for LSD than mature myeloid cells due to their greater longevity and increased capacity to seed tissues. The ability of cells derived from these primitive precursors to seed deep within tissues make them excellent candidates for both cellular therapy and gene transfer techniques to cure a wide range of metabolic diseases.