Combined pancreatic islet and kidney transplantation in a child with unstable type 1 diabetes and end-stage renal disease.

Islet transplantation after successful kidney transplantation is a recognized treatment for adults with diabetes and end-stage renal disease (ESRD), but has not been considered an option in the pediatric population. To our knowledge, we report the first combined islet and kidney transplant in a child. The patient was born with bilateral renal hypoplasia and was diagnosed with type 1 diabetes mellitus at age 13 months. He had erratic glycemic control and hypoglycemia unawareness. At 6 years of age, the child safely underwent simultaneous islet and live donor kidney transplantation. Although function of the islet graft was transient, the combined transplant provided significant benefits in terms of glucose control and overall growth and development. Such an approach represents a viable treatment option for pediatric patients with ESRD and unstable diabetes.

Teplizumab treatment may improve C-peptide responses in participants with type 1 diabetes after the new-onset period: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from a chronic autoimmune process continuing for years after presentation. We tested whether treatment with teplizumab (a Fc receptor non-binding anti-CD3 monoclonal antibody), after the new-onset period, affects the decline in C-peptide production in individuals with type 1 diabetes. METHODS: In a randomised placebo-controlled trial we treated 58 participants with type 1 diabetes for 4-12 months with teplizumab or placebo at four academic centres in the USA. A central randomisation centre used computer generated tables to allocate treatments. Investigators, patients, and caregivers were blinded to group assignment. The primary outcome was a comparison of C-peptide responses to a mixed meal after 1 year. We explored modification of treatment effects in subgroups of patients. RESULTS: Thirty-four and 29 subjects were randomized to the drug and placebo treated groups, respectively. Thirty-one and 27, respectively, were analysed. Although the primary outcome analysis showed a 21.7% higher C-peptide response in the teplizumab-treated group (0.45 vs 0.371; difference, 0.059 [95% CI 0.006, 0.115] nmol/l) (p = 0.03), when corrected for baseline imbalances in HbA(1c) levels, the C-peptide levels in the teplizumab-treated group were 17.7% higher (0.44 vs 0.378; difference, 0.049 [95% CI 0, 0.108] nmol/l, p = 0.09). A greater proportion of placebo-treated participants lost detectable C-peptide responses at 12 months (p = 0.03). The teplizumab group required less exogenous insulin (p < 0.001) but treatment differences in HbA(1c) levels were not observed. Teplizumab was well tolerated. A subgroup analysis showed that treatment benefits were larger in younger individuals and those with HbA(1c) <6.5% at entry. Clinical responders to teplizumab had an increase in circulating CD8 central memory cells 2 months after enrolment compared with non-responders. CONCLUSIONS/INTERPRETATIONS: This study suggests that deterioration in insulin secretion may be affected by immune therapy with teplizumab after the new-onset period but the magnitude of the effect is less than during the new-onset period. Our studies identify characteristics of patients most likely to respond to this immune therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00378508 FUNDING: This work was supported by grants 2007-502, 2007-1059 and 2006-351 from the JDRF and grants R01 DK057846, P30 DK20495, UL1 RR024139, UL1RR025780, UL1 RR024131 and UL1 RR024134 from the NIH.

Peptide mapping and characterisation of glycation patterns of the glima 38 antigen recognised by autoantibodies in Type I diabetic patients.

AIMS/HYPOTHESIS: Glima 38 is an N-glycated neuroendocrine membrane protein of M(r) 38,000, which is recognised by autoantibodies in approximately 20% of patients with Type I (insulin-dependent) diabetes mellitus. The aim of this study was to characterise the carbohydrate moiety and generate peptide maps of glima 38. METHODS: Sera of high immunoreactivity to glima 38 were used to isolate 35-S methionine-labelled protein from betaTC-3 cells and a neuronal cell line GT1.7. Tunicamycin was used to inhibit N-glycation of glima 38 and define the core protein. The carbohydrate moiety was characterised for tunicamycin sensitivity, lectin binding and susceptibility to different endoglycosidases. The protein moiety was subjected to digestion by proteases to define peptide maps. RESULTS: The autoreactive epitopes in glima 38 recognised by Type I diabetic sera are conformational and independent of the carbohydrate moiety. Inhibition of N-glycation of glima 38 in vivo, shows a protein core of M(r) 22,000 in both pancreatic beta-(betaTC3) and neuronal (GT1.7) cell lines. The carbohydrate moieties in the two cell types are distinct but contain a similar amount of terminal sialic acid residues and at least five oligosaccharide chains Glima 38 binds Triticum vulgare and Ricinus communis I lectins. Endoproteinase treatment of the M(r) 22,000 core protein results in peptides of M(r) 4500 and M(r) 20,000 with Lys-C, and peptides of M(r) 4000 and M(r) 11,000-12,000 with Glu-C/V8 and Asp-N proteases. CONCLUSION/INTERPRETATION: The biochemical properties of glima 38 define it as a new autoantigen in Type I diabetes and provide a basis for its purification.

Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass.

Transforming growth factor-beta (TGF-beta) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. To explore the role of endogenous TGF-(beta) in osteoblast function in vivo, we have inhibited osteoblastic responsiveness to TGF-beta in transgenic mice by expressing a cytoplasmically truncated type II TGF-beta receptor from the osteocalcin promoter. These transgenic mice develop an age-dependent increase in trabecular bone mass, which progresses up to the age of 6 months, due to an imbalance between bone formation and resorption during bone remodeling. Since the rate of osteoblastic bone formation was not altered, their increased trabecular bone mass is likely due to decreased bone resorption by osteoclasts. Accordingly, direct evidence of reduced osteoclast activity was found in transgenic mouse skulls, which had less cavitation and fewer mature osteoclasts relative to skulls of wild-type mice. These bone remodeling defects resulted in altered biomechanical properties. The femurs of transgenic mice were tougher, and their vertebral bodies were stiffer and stronger than those of wild-type mice. Lastly, osteocyte density was decreased in transgenic mice, suggesting that TGF-beta signaling in osteoblasts is required for normal osteoblast differentiation in vivo. Our results demonstrate that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties.

Smooth muscle expresses bone morphogenetic protein (Vgr-1/BMP-6) in human fetal intestine.

During human fetal development, autocrine TGF-beta1 regulates the synthesis of specific collagen types by intestinal smooth muscle cells in an age-dependent manner. Vgr-1/BMP6, a member of the TGF-beta superfamily, modulates epithelial, endochondral and neural tissue development in mice: a related peptide is essential to gut morphogenesis in Drosophila. This is the first study to detect vgr-1/BMP-6 during human intestinal organogenesis. Polyclonal antibodies to the precursor and mature fragments of vgr-1 were used in immunohistochemical studies of human intestine at 15, 19 and 24 weeks' gestation. Immunoreactivity was detected with the antibody directed against the precursor portion of vgr-1. Only smooth muscle structures stained for vgr-1 including muscularis propria, muscularis mucosa and vasculature. BMP-6 mRNA was detected by RNase protection assay in cultured muscle cells from 11, 17 and 22 weeks' gestation. This study demonstrates vgr-1/BMP 6 expression in the developing human fetal intestine, exclusively in muscle.

Neonatal hyperinsulinism.

Hyperinsulinism is the most frequent cause of severe, persistent hypoglycemia in neonates and young infants. Timely diagnosis and aggressive treatment are necessary to prevent long-term neurologic sequelae. This article explores the latest advances in the understanding of the pathophysiology of this disorder at the molecular and cellular level. The clinical features, hallmarks for diagnosis, and various treatment options are discussed.

Long-term outcome in children and adolescents after transsphenoidal surgery for Cushing's disease.

Cushing's disease refers specifically to an ACTH-producing pituitary adenoma that stimulates excess cortisol production. Transsphenoidal surgery is the treatment of choice in children and adolescents, but disparate cure rates have been reported, ranging from 50-98%. The discrepancies in cure rate are due primarily to the technical success of the surgery and the length and method of follow-up. We studied 42 consecutive children and adolescents (age, < or = 18 yr) who underwent transsphenoidal exploration for the primary treatment of Cushing's disease at University of California-San Francisco from 1974-1993. Only 7 patients had persistent disease, defined as evidence of Cushing's disease within 6 months of surgery, yielding an initial remission rate of 83%. We comprehensively evaluated 26 of the 35 patients who experienced an initial remission, including testing of the ACTH-adrenocortical axis. The mean duration of follow-up is 7.2 yr (range, 1.5-13.6 yr). Seven experienced a relapse of Cushing's disease, yielding a net remission rate of 73%. Relapses occurred an average of 4.2 yr postoperatively (range, 0.75-6.2 yr). Five patients experienced relapse within 5 yr of surgery, whereas 2 relapsed more than 5 yr postoperatively. Repeat transsphenoidal surgery was performed in 8 patients with persistent or recurrent disease, and 6 of these remain in remission. Low serum or urinary cortisol measurements within the first post-operative week predicted remission of Cushing's disease, but were not necessarily predictive of long-term cure. Hypercortisolism had significant effects on bone metabolism, as reflected by both diminished bone density in the majority of patients examined and decreased growth rate. Both parameters improved after surgical care, although they did not fully normalize. We conclude that transsphenoidal surgery is a safe and effective treatment for pediatric Cushing's disease, but long-term surveillance is necessary to detect possible recurrences.

Idiopathic hypothalamic diabetes insipidus, pituitary stalk thickening, and the occult intracranial germinoma in children and adolescents.

We report nine consecutive children and adolescents [five females and four males; aged 2 yr 8 months (m) to 18 yr 1 m] studied over the last 5 yr with idiopathic central diabetes insipidus. In addition to vasopressin deficiency, anterior pituitary hormone deficiencies were detected, either on evaluation at presentation or during follow-up studies over the following 3 yr. Four patients had an increased concentration of plasma PRL. One patient had multiple pituitary hormone deficiencies at diagnosis, and two others developed the same by 21 m of follow-up. Brain magnestic resonance imaging scans, performed at presentation, were originally interpreted as normal in four of nine patients, except for absence of the bright posterior pituitary signal; after retrospective review, two of nine were considered normal. All of the brain magnetic resonance imaging (MRI) scans showed positive findings by 14 m of follow-up. The first abnormal finding in all patients was isolated pituitary stalk thickening. Evaluation of cerebrospinal fluid (CSF) for hCG was positive in three of eight evaluated patients; the three positive CSF values were found at presentation and 3 and 9 m after presentation. All eight patients assessed were negative for CSF alpha-fetoprotein and cytology, and no patient had serum tumor markers. Transsphenoidal biopsy of the lesion in seven of nine patients showed a germinoma in six patients and inflammatory cells in one. The six patients with documented germinoma comprise 31% of the intracranial germinomas diagnosed in this age group at the University of California-San Francisco during the last 5 yr. The patient with mononuclear inflammatory cells on biopsy along with one other patient have had spontaneous resolution of their stalk thickening. So-called "idiopathic" central diabetes insipidus warrants close follow-up to determine the etiology, especially if anterior pituitary hormone deficiencies are detected. Normal brain MRI scans or scans that show isolated pituitary stalk thickening merit follow-up with serial contrast enhanced brain MRI for the early detection of an evolving occult hypothalamic-stalk lesion. CSF evaluation is recommended at presentation because elevated CSF hCG may precede MRI abnormalities.

Immunoreactive inhibin, müllerian inhibitory substance, and activin as biochemical markers for juvenile granulosa cell tumors.

Ovarian juvenile granulosa cell tumors are a rare cause of sexual precocity. Clinical examination, serum estradiol levels, and pelvic imaging studies have been used traditionally to detect such tumors. Immunoassays for müllerian inhibitory substance and inhibin have recently been noted to provide a more sensitive means of tumor detection in adults. We now describe two girls with this type of tumor in whom serum concentrations of inhibin and müllerian inhibitory substance were used as tumor markers.

A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element?

Complement component C4 is encoded by two nearly identical genes, C4A and C4B, that encode a C4 precursor that is proteolytically cleaved into the alpha, beta and gamma subunits of the mature protein. C4 is expressed primarily in liver and to a much lesser extent in immune cells. We have identified a unique 1 kb RNA transcript, termed Z, that arises from a cryptic promoter lying in the intron between exons 35 and 36 of the C4 gene. Primer extension, RNase protection, and 5' RACE experiments locate the cap site in intron 35, 55 bases upstream from exon 36. Northern blotting and RNase protection assays show that expression of this 1 kb Z RNA transcript is confined to the adrenal gland. Z RNA contains the same open reading frame as C4 which predicts a protein of 131 amino acids, but antisera to C4 do not interact with epitopes on this protein when it is synthesized by cell-free translation, hence the presence or absence of a Z protein in vivo could not be determined. Transfection of Z promoter/reporter constructs into human adrenal NCI-H295 cells shows that most if not all of the sequences required for high-level adrenal expression lie within 235 bases upstream from the cap site, but that this region is inactive when transfected into COS-1, JEG-3 and Hep-G2 cells, suggesting it contains an adrenal-specific element. The 222 bases upstream from the cap site are 75% identical in the human C4A and mouse Slp genes, and contain a potential binding site for steroidogenic factor 1 (SF-1), an orphan zinc-finger nuclear receptor. We propose that this region, like a nearby region in the mouse genome, functions as an upstream element of the P450c21 promoter, and may be a component of an adrenal-specific locus-control region.

Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells.

The transforming growth factor-beta (TGF-beta) superfamily is a group of secreted growth factors that appears to play a central role in mesenchymal differentiation, including cartilage and bone formation. The present study examines the role of one member of this family, vgr-1, also called bone morphogenetic protein-6, in mesenchymal cell differentiation. This factor may be considered as a prototype for the largest subgroup of related factors within the TGF-beta superfamily, the function of which has as yet been poorly defined. vgr-1 has been localized previously to hypertrophic cartilage and has been shown to induce endochondral bone formation in vivo. To further characterize the role of vgr-1 in bone and cartilage differentiation, we stably transfected the pluripotent mesenchymal cell line ROB-C26 with a vector to overexpress vgr-1. Overexpression of this factor did not affect cell shape or morphology, but it enhanced osteoblastic differentiation in vitro and altered cellular responsiveness to retinoic acid. Furthermore, the extracellular matrix produced by these vgr-1-overexpressing cells induced ectopic bone formation in vivo and osteoblastic differentiation in vitro, similar to the matrix produced by C26 cells treated with retinoic acid. The osteoinductive effect of the matrix from vgr-1-overexpressing cells was blocked using a neutralizing vgr-1 antibody but not with a neutralizing TGF-beta 1 antibody, indicating that vgr-1 alone was required for this osteogenic effect. In contrast, the osteoinductive effect of matrix from retinoic acid-treated cells was blocked with both vgr-1 and TGF-beta 1 antibodies, suggesting that TGF-beta 1 may act prior to vgr-1 during osteoblastic differentiation. We further demonstrated that osteoinduction by vgr-1 was dependent on presentation of vgr-1 within the matrix, because the osteoinductive effect of matrix from vgr-1-overexpressing cells could not be mimicked with the addition of soluble vgr-1 to parental C26 cells. Finally, overexpression of MyoD within the C26 cells overexpressing vgr-1 converted the cells to myoblasts, indicating that vgr-1 had induced early osteoblastic.

Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo.

Members of the TGF-beta superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-beta-related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-1-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endochondral bone formation.

Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B.

A human gene termed XB overlaps the P450c21B gene encoding steroid 21-hydroxylase and encodes a protein that closely resembles extracellular matrix proteins. Sequencing of phage and cosmid clones and of cDNA fragments shows that the XB gene spans 65 kb of DNA, consisting of 39 exons that encode a 12-kb mRNA. The predicted protein of over 400 kD consists of five distinct domains: a signal peptide, a hydrophobic domain containing three heptad repeats, a series of 18.5 EGF-like repeats, 29 fibronectin type III repeats, and a carboxy-terminal fibrinogen-like domain. Because the structure of the protein encoded by the XB gene closely resembles tenascin, we term this protein tenascin-X (TN-X), and propose a simplified nomenclature system for the family of tenascins. RNase protection experiments show that the TN-X transcript is expressed ubiquitously in human fetal tissues, with the greatest expression in the fetal testis and in fetal skeletal, cardiac, and smooth muscle. Two adrenal-specific transcripts, P450c21B (steroid 21-hydroxylase) and Y (an untranslated transcript) overlap the XB gene on the complementary strand of DNA, yielding a unique array of overlapping transcripts: a "polygene." In situ hybridization histochemistry experiments show that the TN-X transcript and the P450c21 and Y transcripts encoded on the complementary DNA strand are all expressed in the same cells of the human adrenal cortex. Genetic data suggest that TN-X may be essential for life.

Mental retardation locus in Xp21 chromosome microdeletion.

Xp21 microdeletion syndrome is associated with variable size Xp21 deletions that usually include the glycerol kinase locus. The clinical phenotypes we studied in this chromosome region include: Xpter - Aland Island eye disease (AIED) -adrenal hypoplasia (AH) -glycerol kinase (GKD) -Duchenne muscular dystrophy (DMD) -retinitis pigmentosa (RP) -ornithine transcarbamylase (OTC) -centromere. In a compilation of 18 individuals in 14 families with the AH, GKD, and DMD loci deleted, 17 were male and all were developmentally delayed. In contrast, we report mentally retarded female carriers in two Xp21 deletion syndrome families with DMD, GKD, and AH in affected males. In the first family with normal karyotypes, a submicroscopic deletion was associated with DMD in the retarded male and with retardation in carrier females. In the second family an X chromosome with a cytogenetically deleted Xp21 distal to the OTC and RP genes segregated in the affected male and retarded female carriers. DNA analysis at the DMD locus verified the cytogenetic findings. This report of mental retardation in otherwise asymptomatic female carriers of Xp21 deletion classifies one form of mental retardation in females.

Abundant adrenal-specific transcription of the human P450c21A "pseudogene".

Human adrenal steroid 21-hydroxylase (P450c21) is encoded by the CYP21A1 (21B) gene located in the class III region of the HLA locus. A tandemly duplicated gene designated CYP21A1P (21A), which lies 30 kilobases upstream, contains several point mutations and an 8-base pair deletion so that it cannot encode P450c21 protein; as a result, it is generally considered to be a pseudogene. We previously showed that two additional genes, XA and XB, lie on the opposite strand of DNA overlapping the 3'-ends of the 21A and 21B genes. We have now identified a third pair of duplicated overlapping genes in this locus, termed YA and YB, whose transcriptional orientation is the same as 21A and 21B and opposite to XA and XB. YA transcripts use the 21A promoter, have 5'-ends that are similar to 21B mRNA, and have approximately 10-20% of the abundance of 21B transcripts, but have unique 3'-ends. The YA gene encodes a 7.5-kilobase RNA that overlaps XA completely and a 3.0-kilobase RNA that excludes most of XA. The YB gene appears to be similar in size and organization to YA. The YA and YB genes extend beyond the limit of the duplication in this locus; hence, their cDNAs are distinguishable by differences in their 3'-sequences. YA and YB transcripts are expressed only in the fetal and adult adrenal glands, but their cDNAs do not contain a long open reading frame. Although the function of these genes is not yet clear, the complex genetic organization of three overlapping genes (21/X/Y) appears to be unique among higher eukaryotes. As YA transcription is initiated by the 21A 5'-flanking DNA and includes 21A sequences, the designation of 21A as a "pseudogene" merits reconsideration.

Congenital lipoid adrenal hyperplasia--genes for P450scc, side chain cleavage enzyme, are normal.

In the most severe form of congenital adrenal hyperplasia (CAH), termed lipoid CAH, both the adrenals and gonads fail to convert cholesterol to pregnenolone, so that no steroid hormones are made. Newborns have female external genitalia irrespective of karyotype, and suffer a severe salt-losing form of CAH. Previous studies have shown that adrenal or gonadal mitochondria from these patients also fail to convert cholesterol to pregnenolone in vitro, implicating a lesion in the single gene for P450scc, which is the sole enzyme converting cholesterol to pregnenolone. Two patients with XY karyotypes had female genitalia and unmeasurable steroids after stimulation with ACTH and hCG. ACTH stimulation tests of parents, obligate heterozygotes, showed normal stimulation of all precursor steroids. Southern blotting patterns of the P450scc gene were normal. Oligonucleotide-initiated enzymatic amplification (PCR) of all P450scc exons showed normal sequences on multiple amplifications and sequencing reactions, indicating normal P450scc genes. Northern blots of testicular RNA from a 6-month-old patient and from a control fetus showed normal P450scc mRNA, indicating a normal P450scc promoter. Reprobing of the blot with our cloned human cDNAs for adrenodoxin reductase and adrenodoxin showed that these electron transport cofactors used by P450scc were also normal. Similarly, probing with cDNAs for all three known factors involved in cholesterol transport to the mitochondria-sterol carrier protein 2, endozepine, and steroidogenesis activator peptide were also normal. These results suggest that the lesion in lipoid CAH is not in the P450scc system or in any known step upstream from P450scc.

Mechanism and consequences of the duplication of the human C4/P450c21/gene X locus.

The adjacent C4 and P450c21 genes encode the fourth component of serum complement and steroid 21-hydroxylase respectively, and are tandemly duplicated in the human, murine, and bovine genomes. We recently cloned a cDNA for another duplicated gene, operationally termed X, which overlaps the 3' end of human P450c21 and has the opposite transcriptional orientation. Thus, the organization of the locus is 5'-C4A-21A-XA-C4B-21B-XB-3' (Y. Morel, J. Bristow, S. E. Gitelman, and W. L. Miller, Proc. Natl. Acad. Sci. USA 86:6582-6586, 1989). To determine how this locus was duplicated, we sequenced the DNA at the duplication boundaries and the 7 kb between P450c21A and C4B comprising the XA locus. The sequences located the duplication boundaries precisely and indicate that the duplication occurred by nonhomologous recombination. The boundaries are substantially different from those of the corresponding duplication in the mouse genome, suggesting that similar gene duplications may have occurred independently in ancestors of rodents and primates after mammalian speciation. Compared with XB, the XA gene is truncated at its 5' end and bears a 121-bp intragenic deletion causing a frameshift and premature translational stop signal. Nevertheless, XA is transcribed into a stable 2.6-kb polyadenylated RNA that is expressed uniquely in the adrenal gland.