Growth parameters in children with achondroplasia: A 7-year, prospective, multinational, observational study.

PURPOSE: This study was undertaken to collect baseline growth parameters in children with achondroplasia who might enroll in interventional trials of vosoritide, and to establish a historical control. METHODS: In this prospective, observational study, participants (≤17 years) underwent a detailed medical history and physical examination and were followed every 3 months until they finished participating in the study by enrolling in an interventional trial or withdrawing. RESULTS: A total of 363 children were enrolled (28 centers, 8 countries). Mean (SD) follow up was 20.4 (15.0) months. In participants <1 year, mean annualized growth velocity (AGV) was 11.6 cm/year for girls and 14.6 cm/year for boys. By age 1 year, mean AGV decreased to 7.4 cm/year in girls and 7.1 cm/year in boys. By age 10 years, mean AGV decreased to 3.6 cm/year for both sexes. Mean height z-score in participants <1 year was -2.5 for girls and -3.2 for boys and decreased up to the age 5 years (-5.3 for girls; -4.6 for boys). Girls and boys had a disproportionate upper-to-lower body segment ratio. Mean ratio was highest in participants aged <1 year (2.9 for girls; 2.8 for boys) and decreased gradually to approximately 2 in both sexes from 4 years of age onward. CONCLUSION: This study represents one of the largest datasets of prospectively collected medical and longitudinal growth data in children with achondroplasia. It serves as a robust historical control to measure therapeutic interventions against and to further delineate the natural history of this condition.

De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids.

Hematopoietic humanized (hu) mice are powerful tools for modeling the action of human immune system and are widely used for preclinical studies and drug discovery. However, generating a functional human T cell compartment in hu mice remains challenging, primarily due to the species-related differences between human and mouse thymus. While engrafting human fetal thymic tissues can support robust T cell development in hu mice, tissue scarcity and ethical concerns limit their wide use. Here, we describe the tissue engineering of human thymus organoids from inducible pluripotent stem cells (iPSC-thymus) that can support the de novo generation of a diverse population of functional human T cells. T cells of iPSC-thymus-engrafted hu mice could mediate both cellular and humoral immune responses, including mounting robust proinflammatory responses on T cell receptor engagement, inhibiting allogeneic tumor graft growth and facilitating efficient Ig class switching. Our findings indicate that hu mice engrafted with iPSC-thymus can serve as a new animal model to study human T cell-mediated immunity and accelerate the translation of findings from animal studies into the clinic.

Construction of Thymus Organoids from Decellularized Thymus Scaffolds.

One of the hallmarks of modern medicine is the development of therapeutics that can modulate immune responses, especially the adaptive arm of immunity, for disease intervention and prevention. While tremendous progress has been made in the past decades, manipulating the thymus, the primary lymphoid organ responsible for the development and education of T lymphocytes, remains a challenge. One of the major obstacles is the difficulty to reproduce its unique extracellular matrix (ECM) microenvironment that is essential for maintaining the function and survival of thymic epithelial cells (TECs), the predominant population of cells in the thymic stroma. Here, we describe the construction of functional thymus organoids from decellularized thymus scaffolds repopulated with isolated TECs. Thymus decellularization was achieved by freeze-thaw cycles to induce intracellular ice crystal formation, followed by detergent-induced cell lysis. Cellular debris was removed with extensive wash. The decellularized thymus scaffolds can largely retain the 3D extracellular matrix (ECM) microenvironment that can support the recolonization of TECs. When transplanted into athymic nude mice, the reconstructed thymus organoids can effectively promote the homing of bone marrow-derived lymphocyte progenitors and support the development of a diverse and functional T cell repertoire. Bioengineering of thymus organoids can be a promising approach to rejuvenate/modulate the function of T-cell mediated adaptive immunity in regenerative medicine.

Restoration of Thymus Function with Bioengineered Thymus Organoids.

The thymus is the primary site for the generation of a diverse repertoire of T-cells that are essential to the efficient function of adaptive immunity. Numerous factors varying from aging, chemotherapy, radiation exposure, virus infection and inflammation contribute to thymus involution, a phenomenon manifested as loss of thymus cellularity, increased stromal fibrosis and diminished naïve T-cell output. Rejuvenating thymus function is a challenging task since it has limited regenerative capability and we still do not know how to successfully propagate thymic epithelial cells (TECs), the predominant population of the thymic stromal cells making up the thymic microenvironment. Here, we will discuss recent advances in thymus regeneration and the prospects of applying bioengineered artificial thymus organoids in regenerative medicine and solid organ transplantation.

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor-Specific Immune Tolerance to Allografts.

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine.

Bioengineering mini functional thymic units with EAK16-II/EAKIIH6 self-assembling hydrogel.

Herein, we highlight the technical feasibility of generating a functional mini thymus with a novel hydrogel system, based on a peptide-based self-assembly platform that can induce the formation of 3-D thymic epithelial cell (TEC) clusters. Amphiphilic peptide EAK16-II co-assembled with its histidinylated analogue EAKIIH6 into beta-sheet fibrils. When adaptor complexes (recombinant protein A/G molecules loaded with both anti-His and anti-EpCAM IgGs) were added to the mix, TECs were tethered to the hydrogel and formed 3-D mini clusters. TECs bound to the hydrogel composites retained their molecular properties; and when transplanted into athymic nude mice, they supported the development of functional T-cells. These mini thymic units of TECs can be useful in clinical applications to reconstitute T-cell adaptive immunity.

Compromised central tolerance of ICA69 induces multiple organ autoimmunity.

For reasons not fully understood, patients with an organ-specific autoimmune disease have increased risks of developing autoimmune responses against other organs/tissues. We identified ICA69, a known ß-cell autoantigen in Type 1 diabetes, as a potential common target in multi-organ autoimmunity. NOD mice immunized with ICA69 polypeptides exhibited exacerbated inflammation not only in the islets, but also in the salivary glands. To further investigate ICA69 autoimmunity, two genetically modified mouse lines were generated to modulate thymic ICA69 expression: the heterozygous ICA69(del/wt) line and the thymic medullary epithelial cell-specific deletion Aire-ΔICA69 line. Suboptimal central negative selection of ICA69-reactive T-cells was observed in both lines. Aire-ΔICA69 mice spontaneously developed coincident autoimmune responses to the pancreas, the salivary glands, the thyroid, and the stomach. Our findings establish a direct link between compromised thymic ICA69 expression and autoimmunity against multiple ICA69-expressing organs, and identify a potential novel mechanism for the development of multi-organ autoimmune diseases.

Evolution of hepatic steatosis to fibrosis and adenoma formation in liver-specific growth hormone receptor knockout mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver diseases closely associated with obesity and insulin resistance; deficient growth hormone (GH) action in liver has been implicated as a mechanism. Here, we investigated the evolution of NAFLD in aged mice with liver-specific GHR deletion. METHODS: We examined glucose tolerance, insulin responsiveness, and lipid profiles in aged male mice (44-50 weeks) with GHRLD. We performed proteomics analysis, pathway-based Superarray assay, as well as quantitative RT-PCR to gain molecular insight into the mechanism(s) of GHR-deficiency-mediated NAFLD. In addition, we examined the pathological changes of livers of aged GHRLD male mice. RESULTS: The biochemical profile was consistent with that of the metabolic syndrome: abnormal glucose tolerance, impaired insulin secretion, and hyperlipidemia. RT-qPCR analysis of key markers of inflammation revealed a three- to fivefold increase in TNFα and CCL3, confirming the presence of inflammation. Expression of fibrotic markers (e.g., Col1A2 and Col3A1) was significantly increased, together with a two- to threefold increase in TGFß transcripts. Proteomics analyses showed a marked decrease of Mup1 and Selenbp2. In addition, pathway-analysis showed that the expression of cell cycle and growth relevant genes (i.e., Ccnd1, Socs2, Socs3, and Egfr) were markedly affected in GHRLD liver. Microscopic analyses (H&E) of GHRLD livers revealed the presence of hepatic adenomas of different stages of malignancy. CONCLUSION: Abrogation of GH signaling in male liver leads to metabolic syndrome, hepatic steatosis, increased inflammation and fibrosis, and development of hepatic tumor. Since obesity, a common precursor of NAFLD, is a state of deficient GH secretion and action, the GHRLD model could be used to unravel the contribution of compromised hepatic GH signaling in these pathological processes, and help to identify potential targets for intervention.

Mutational analyses of the ATP6V1B1 and ATP6V0A4 genes in patients with primary distal renal tubular acidosis.

BACKGROUND: Mutations in the ATP6V1B1 and the ATP6V0A4 genes cause primary autosomal-recessive distal renal tubular acidosis (dRTA). Large deletions of either gene in patients with dRTA have not been described. METHODS: The ATP6V1B1 and ATP6V0A4 genes were directly sequenced in 11 Japanese patients with primary dRTA from nine unrelated kindreds. Large heterozygous deletions were analyzed by quantitative real-time polymerase chain reaction (PCR). The clinical features of the 11 patients were also investigated. RESULTS: Novel mutations in the ATP6V1B1 gene were identified in two kindreds, including frameshift, in-frame insertion and nonsense mutations. Large deletions in the ATP6V0A4 gene were identified in two kindreds. Exon 15 of ATP6V0A4 was not amplified in one patient, with a long PCR confirming compound heterozygous deletions of 3.7- and 6.9-kb nucleotides, including all of exon 15. Direct DNA sequencing revealed a heterozygous frameshift mutation in ATP6V0A4 in another patient, with quantitative real-time PCR indicating that all exons up to exon 8 were deleted in one allele. Clinical investigation showed that four of the six patients with available clinical data presented with hyperammonemia at onset. CONCLUSIONS: To our knowledge, these dRTA patients are the first to show large deletions involving one or more entire exons of the ATP6V0A4 gene. Quantitative PCR amplification may be useful in detecting heterozygous large deletions. These results expand the spectrum of mutations in the ATP6V0A4 and ATP6V1B1 genes associated with primary dRTA and provide insight into possible structure-function relationships.

Diagnostic usefulness of 3 tesla MRI of the brain for cushing disease in a child.

It is sometimes difficult to confirm the location of a microadenoma in Cushing disease. Recently, we experienced an 11-yr-old female case of Cushing disease with hyperprolactinemia. She was referred to our hospital because of decrease of height velocity with body weight gain. On admission, she had typical symptoms of Cushing syndrome. Although no pituitary microadenomas were detected on 1.5 Tesla MRI of the brain, endocrinological examinations including IPS and CS sampling were consistent with Cushing disease with hyperprolactinemia. Oral administration of methyrapone instead of neurosurgery was started after discharge, but subsequent 3 Tesla MRI of the brain clearly demonstrated a 3-mm less-enhanced lesion in the left side of the pituitary gland. Finally, transsphenoidal surgery was performed, and a 3.5-mm left-sided microadenoma was resected. Compared with 1.5 Tesla MRI, 3 Tesla MRI offers the advantage of a higher signal to noise ratio (SNR), which provides higher resolution and proper image quality. Therefore, 3 Tesla MRI is a very useful tool to localize microadenomas in Cushing disease in children as well as in adults. It will be the first choice of radiological examinations in suspected cases of Cushing disease.

Gaucher disease patient with myoclonus epilepsy and a novel mutation.

The N188S mutation in Gaucher disease is associated with myoclonus epilepsy. We performed genetic analysis on a patient with progressive myoclonus epilepsy, who had received antiepileptic drugs for over 10 years. We detected N188S/G199D on the gene encoding glucocerebrosidase. Mutant proteins carrying each mutation were expressed in COS-1 cells (a commonly used cell line which derives from kidney cells of the African green monkey). Measurements of enzymatic activity and Western blotting analysis were performed. When residual activities were measured, glucocerebrosidase with the N188S mutation exhibited 50% activity of the wild type, and with G199D, 7.4%. Neither mutation influenced the stability of the enzyme protein. These data suggested a diagnosis of Gaucher disease for this patient, and indicated that G199D is a novel mutation.

Clinical and genetic study of Japanese patients with type 3 Gaucher disease.

Information on the phenotypic variations seen in patients with type 3 (chronic neuronopathic) Gaucher disease (GD) is still limited compared with type 1 GD. We retrospectively investigated the clinical features of 42 Japanese patients with type 3 GD. The 42 patients classified as type 3 fell into two groups: those diagnosed as having type 3 GD at diagnosis (group A; n = 24) and those thought to have type 1 at diagnosis but who later developed neurological symptoms (group B; n = 18). The genotype of group A patients varied widely; however, L444P/L444P and L444P/F213I genotypes accounted for 83% in group B. All the patients who did not receive enzyme replacement with alglucerase or imiglucerase (4 in group A, 2 in group B) died. Nineteen patients received enzyme replacement in group A; however, 7 of these died despite the therapy. On the other hand, 14 patients received enzyme replacement alone in group B and 13 of them survived. Among the ERT-treated patients who survived, only one of 12 in group A and 12 out of 13 in group B can walk unaided. In conclusion, some Japanese GD patients who are thought to have type 1 at diagnosis develop neurological symptoms during their clinical course, and careful observation is essential for patients with characteristic genotypes. Moreover, enzyme replacement alone might not have a sufficient effect on the early onset neurological symptoms in type 3 patients. A different treatment strategy is needed to improve the prognosis of these patients.

Roles of specific cytokines in bone remodeling and hematopoiesis in Gaucher disease.

BACKGROUND: Gaucher disease type 1 and type 3 are characterized by bone disease and hematological symptoms. It is known that monocyte/macrophage lineage is activated in Gaucher disease, and accordingly certain cytokines are elevated in blood. The aim of the present study was to explore the possible relationships between cytokines and bone remodeling and hematological abnormalities in this disease. METHODS: The concentrations of seven cytokines and two related proteins were measured in patients with Gaucher disease type 1 and type 3 (n= 8; age range, 2-50 years) who had received enzyme replacement therapy. RESULTS: Concentrations of interleukin-18 and transforming growth factor-beta1 were elevated in patients of all clinical types. Elevation of these cytokines in Gaucher disease has not been previously reported. Analysis of correlation among cytokines and bone-turnover markers showed that interleukin-18 concentration was correlated with each of two bone formation markers of bone-specific alkaline phosphatase activity and osteocalcin concentration, whereas macrophage colony-stimulating factor concentration correlated with the bone absorption marker of N-telopeptide to helix in urine. Concentrations of macrophage colony-stimulating factor and tumor necrosis factor-alpha were inversely correlated with hemoglobin concentration. CONCLUSIONS: Interleukin-18 and monocyte macrophage colony-stimulating factor are cytokines mainly involved in the mechanism of bone disease, while macrophage colony-stimulating factor and tumor necrosis factor-alpha may play a role in the development of hematological abnormalities in Gaucher disease.

Successful intrauterine therapy for fetal goitrous hypothyroidism during late gestation.

We experienced a case of fetal goitrous hypothyroidism in an infant delivered by a 33-year-old woman receiving 300 mg/day of propylthiouracil (PTU) for hyperthyroidism due to Graves' disease. A large fetal goiter (maximum diameter, 60 mm) was detected by magnetic resonance imaging (MRI) at 36 weeks of gestation. Initial fetal blood sampling revealed hypothyroidism with a serum thyroid-stimulating hormone (TSH) of 99 microIU/mL, free triiodothyronine (T(3)) of 1.97 pg/mL, and free thyroxine (T(4)) of 0.29 ng/dL. Consequently, a diagnosis of fetal goitrous hypothyroidism due to transplacental passage of maternal PTU was made. To reduce the risk of perinatal complications, 300 microg of levothyroxine sodium (L-T(4)) was administered into the maternal amniotic fluid twice between 37 and 38 weeks of gestation. Subsequent fetal MRI showed that the size of goiter had decreased. At 38 weeks and 5 days of gestation, a 3042-g male infant was born by cesarean section. There were no severe complications at delivery, although mild tachypnea was observed and the infant's thyroid gland was slightly enlarged. He was treated with L-T(4) for two weeks. At present, his growth and neurological development are normal. This case indicates that intrauterine therapy by the intraamniotic administration of L-T(4) can be effective in treating fetal goitrous hypothyroidism even during late gestation.

Identification and functional analysis of the novel S179R POU1F1 mutation associated with combined pituitary hormone deficiency.

CONTEXT: The pituitary-specific transcription factor 1 plays a key role in the development and differentiation of three pituitary cell types: somatotrophs, lactotrophs, and thyrotrophs. Several mutations of the human gene (called POU1F1) have been shown to be responsible for a phenotype of combined pituitary hormone deficiency involving GH, prolactin (PRL), and TSH. OBJECTIVE: We have identified a novel homozygous C to G mutation in exon 4 of the POU1F1 gene (S179R) in a patient with this rare phenotype. We analyzed the functional consequences of this S179R mutation associated with a single-amino acid change in the POU-specific domain. METHODS: Consequences of this mutation on transcriptional activities by transfection studies in alphaT3 cells, DNA binding ability by EMSA, structural properties, and nuclear accumulation of POU1F1 were investigated. RESULTS: The transactivation capacity of this mutant was markedly decreased on the GH1, PRL, TSHbeta, and POU1F1 genes. Interestingly, this mutation abolished the functional interaction of POU1F1 on the PRL promoter with the coactivator cAMP response element-binding protein-binding protein but not with the transcription factor LIM homeodomain transcription factor 3. The S179R mutant displayed normal nuclear accumulation but a markedly decreased binding to a DNA response element in keeping with crystallographic data, suggesting that the S179R mutation might interfere with DNA binding. CONCLUSIONS: Together with previous data, our study indicates that both DNA binding and interaction with cofactors like cAMP response element-binding protein-binding protein are critical for POU1F1 function and that functional and structural properties of abnormal POU1F1 proteins are variously influenced by the type of mutations.

A novel mutation of the arginine vasopressin receptor 2 gene in a patient with congenital nephrogenic diabetes insipidus.

We have identified a novel mutation of the arginine vasopressin receptor 2 (AVPR2) gene in a case of congenital X-linked nephrogenic diabetes insipidus (NDI). The patient was a 2-mo-old Japanese boy with persistent fever and failure to thrive. He was diagnosed as having congenital NDI by clinical and laboratory findings. Molecular analysis demonstrated that he was hemizygous for a G to C transversion in exon 2 of the AVPR2 gene which resulted in a glycine to arginine substitution (G107R) at the 107th codon of the first extracellular loop. His mother was heterozygous for the same mutation. We speculated that the G107R mutation would interfere with the binding capacity of the AVPR2, since G107R is located near F105 and R106, both of which are crucial for ligand binding. In cases of X-linked NDI, mutations in the AVPR2 gene are distributed widely. Thus, DNA analysis throughout the gene is of clinical value for the identification of female carriers, and it also gives precise information for genetic counseling.