Milton R. Brown

Mutation of the POU-Specific Domain of Pit-1 and Hypopituitarism Without Pituitary Hypoplasia

A point mutation in the POU-specific portion of the human gene that encodes the tissuespecific POU-domain transcription factor, Pit-1, results in hypopituitarism, with deficiencies of growth hormone, prolactin, and thyroid-stimulating hormone. In two unrelated Dutch families, a mutation in Pit-1 that altered an alanine in the first putative α helix of the POU-specific domain to proline was observed. This mutation generated a protein capable of binding to DNA response elments but unable to effectively activate its known target genes, growth hormone and prolactin. The phenotype of the affected individuals suggests that the mutant Pit-1 protein is competent to initiate other programs of gene activation required for normal proliferation of somatotrope, lactotrope, and thyrotrope cell types. Thus, a mutation in the POU-specific domain of Pit-1 has a selective effect on a subset of Pit-1 target genes.

Dense genotyping of immune-related disease regions identifies 14 new susceptibility loci for juvenile idiopathic arthritis

We used the Immunochip array to analyze 2,816 individuals with juvenile idiopathic arthritis (JIA), comprising the most common subtypes (oligoarticular and rheumatoid factor–negative polyarticular JIA), and 13,056 controls. We confirmed association of 3 known JIA risk loci (the human leukocyte antigen (HLA) region, PTPN22 and PTPN2) and identified 14 loci reaching genome-wide significance (P < 5 × 10−8) for the first time. Eleven additional new regions showed suggestive evidence of association with JIA (P < 1 × 10−6). Dense mapping of loci along with bioinformatics analysis refined the associations to one gene in each of eight regions, highlighting crucial pathways, including the interleukin (IL)-2 pathway, in JIA disease pathogenesis. The entire Immunochip content, the HLA region and the top 27 loci (P < 1 × 10−6) explain an estimated 18, 13 and 6% of the risk of JIA, respectively. In summary, this is the largest collection of JIA cases investigated so far and provides new insight into the genetic basis of this childhood autoimmune disease.

Dual Delivery of Hepatocyte and Vascular Endothelial Growth Factors via a Protease-Degradable Hydrogel Improves Cardiac Function in Rats

Acute myocardial infarction (MI) caused by ischemia and reperfusion (IR) is the most common cause of cardiac dysfunction due to local cell death and a temporally regulated inflammatory response. Current therapeutics are limited by delivery vehicles that do not address spatial and temporal aspects of healing. The aim of this study was to engineer biotherapeutic delivery materials to harness endogenous cell repair to enhance myocardial repair and function. We have previously engineered poly(ethylene glycol) (PEG)-based hydrogels to present cell adhesive motifs and deliver VEGF to promote vascularization in vivo. In the current study, bioactive hydrogels with a protease-degradable crosslinker were loaded with hepatocyte and vascular endothelial growth factors (HGF and VEGF, respectively) and delivered to the infarcted myocardium of rats. Release of both growth factors was accelerated in the presence of collagenase due to hydrogel degradation. When delivered to the border zones following ischemia-reperfusion injury, there was no acute effect on cardiac function as measured by echocardiography. Over time there was a significant increase in angiogenesis, stem cell recruitment, and a decrease in fibrosis in the dual growth factor delivery group that was significant compared with single growth factor therapy. This led to an improvement in chronic function as measured by both invasive hemodynamics and echocardiography. These data demonstrate that dual growth factor release of HGF and VEGF from a bioactive hydrogel has the capacity to significantly improve cardiac remodeling and function following IR injury.

Association of Glutathione-S-Transferase-P1 (GST-P1) Polymorphisms with Bronchopulmonary Dysplasia

OBJECTIVE: Reactive oxygen species (ROS) contribute to oxidative lung injury. The glutathione-S-transferases (GST) family and microsomal epoxide hydrolase (mEPHx) enzymes detoxify ROS, and genetic polymorphisms alter this detoxification. We hypothesized that polymorphisms encoding for less efficient enzymes were associated with bronchopulmonary dysphasia (BPD).STUDY DESIGN: We determined allelic distribution of these polymorphisms in a pilot study of 35 BPD cases and 98 controls. χ2 and regression analysis were performed.RESULTS: GST P1 val105ile distribution differed between the groups, with the more efficient val/val allele predominately in controls (p≤0.05). When controlling for race and sex, BPD cases were less likely to be homozygotes for the val/val isoform (OR 0.21, CI: 0.045–0.95, p=0.04) and more likely to possess the less efficient ile isoform (OR 4.5, CI: 1.0–20.7, p=0.05).CONCLUSIONS: This pilot study suggests that BPD is associated with the presence of the GST-P1 105ile allele. Future prospective studies are warranted.

CENTRAL HYPOTHYROIDISM REVEALS COMPOUND HETEROZYGOUS MUTATIONS IN THE PIT-1 GENE

Mutations in the gene encoding the Pit-1 transcriptional activator interfere with the embryologic determination and ultimate functions of anterior pituitary cells that produce growth hormone (GH), prolactin (Prl) and thyroid-stimulating hormone (TSH). Central hypothyroidism is often the presenting feature of combined pituitary hormone deficiency (CPHD), but it is not detected in screening programs that rely upon elevation of TSH. We report a child whose hypothyroidism was recognized clinically at age 6 weeks, and subsequently found to have GH and Prl as well as TSH deficiency. With thyroxine and GH replacement he has reached the 70th percentile for height and has normal intelligence. Molecular analysis of genomic DNA for Pit-1 revealed the presence of compound heterozygous recessive mutations: a nonsense mutation in codon 172 and a novel missense mutation substituting glycine for glutamate at codon 174. This case is the first demonstration of CPHD due to compound heterozygous Pit-1 point mutations, as most reported cases of the CPHD phenotype involve either the dominant negative R271W allele or homozygosity for recessive Pit-1 mutations. Therefore, in cases of CPHD, the possibilities of compound heterozygosity for two different Pit-1 mutations, or homozygosity for mutations in the epigenetic gene, Prop-1, should be considered.

A Mutational Hot Spot in the Prop-1 Gene in Russian Children with Combined Pituitary Hormone Deficiency

Combined pituitary hormone deficiency (CPHD), including growth hormone (GH), prolactin (Prl) and thyroid-stimulating hormone (TSH) in children is now considered a heterogeneous syndrome. Recent findings on expression of mouse pituitary-specific homeodomain factors demonstrate dependence of adenopituitary ontogeny on interactive expression of these factors, suggesting their involvement in etiology of CPHD. Prophet of Pit-1 (Prop-1) gene, a novel pituitary-specific homeodomain factor, was analyzed in 14 Russian children with CPHD, in whom Pit-1 gene was intact. We found a mutational hot spot in three patients from two families in homeodomain part of the second exon of Prop-1 gene. The common 2- base pair deletion (GA296) in the homozygous state resulted in a Serine to Stop codon (S109X) substitution and generated a truncated Prop-1 protein. Parents were phenotypically normal and heterozygous for GA296 deletion, indicating an autosomal recessive inheritance. These results demonstrate a novel type of Prop-1 gene mutation as one of the causes of CPHD in Russian patients.

Faster pharmacokinetics and increased patient acceptance of intradermal insulin delivery using a single hollow microneedle in children and adolescents with type 1 diabetes

In an effort to improve compliance with insulin therapy and to accelerate insulin pharmacokinetics, we tested the hypothesis that intradermal insulin delivery using a hollow microneedle causes less pain and leads to faster onset and offset of insulin pharmacokinetics in children and adolescents with type 1 diabetes (T1DM) compared with a subcutaneous, insulin pump catheter.

Hoechst-IR: an imaging agent that detects necrotic tissue in vivo by binding extracellular DNA.

Cell necrosis is central to the progression of numerous diseases, and imaging agents that can detect necrotic tissue have great clinical potential. We demonstrate here that a small molecule, termed Hoechst-IR, composed of the DNA binding dye Hoechst and the near-infrared dye IR-786, can image necrotic tissue in vivo via fluorescence imaging. Hoechst-IR detects necrosis by binding extracellular DNA released from necrotic cells and was able to image necrosis generated from a myocardial infarction and lipopolysaccharide/d-galactosamine (LPS-GalN) induced sepsis.

Cultured Human Bone Marrow–Derived CD31+ Cells Are Effective for Cardiac and Vascular Repair Through Enhanced Angiogenic, Adhesion, and Anti-Inflammatory Effects

BACKGROUND Cell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31(+) cells are a promising cell source owing to their high angiovasculogenic and paracrine activities. OBJECTIVES This study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31(+) cells, and to determine whether these cultured CD31(+) cells are effective for cardiac and vascular repair. METHODS CD31(+) cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms. RESULTS The CD31(+) cells cultured in endothelial cell medium (EC-CD31(+) cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31(+) cells. When implanted into mouse MI or HLI models, EC-CD31(+) cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31(+) cells. Histologically, injected EC-CD31(+) cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year. CONCLUSIONS Short-term cultured EC-CD31(+) cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31(+) cells may be a promising source for treating ischemic cardiovascular diseases.

Screening for MEN1 tumor suppressor gene mutations in sporadic pituitary tumors.

The molecular pathogenesis of the majority of sporadic pituitary tumors is largely unknown. Pituitary adenomas can develop sporadically or as a part of multiple endocrine neoplasia type 1 (MEN1). The MEN1 is thought to be a tumor suppressor gene based on loss of heterozygosity (LOH) for polymorphic markers on 11q13 in tumors of the pancreas, parathyroid, and pituitary. Most patients with familial and sporadic MEN1 carry germ-line mutations in the MEN1 gene. Two previous studies and recently a third one have analyzed mutations by sequencing the MEN1 gene in sporadic pituitary tumors but yielded conflicting results. This study was to investigate and clarify the potential role of MEN1 mutations, in sporadic pituitary adenomas. First, we examined 59 sporadic pituitary adenomas by analyzing LOH on 11q13 in the MEN1 minimal interval with microsatellite analysis. We found 3 tumors with LOH in 1 to 4 polymorphic markers in the MEN1 region. Sequencing analysis did not reveal any mutations in the coding region of the MEN1 gene. However, we found 3 polymorphisms, one of which was a novel CAC to CAT transition encoding His433His, in exon 9. The data show that while LOH occurs in some sporadic pituitary tumors, inactivating mutations of the tumor suppressor gene MEN1 are rare. These results also suggest there may be another additional tumor suppressor gene at this locus which is involved in the pathogenesis of sporadic pituitary neoplasms.