Chanika Phornphutkul

Towards a vaccine for rheumatic fever: identification of a conserved target epitope on M protein of group A streptococci

Rheumatic fever and rheumatic heart disease remain very common in developing countries, and a vaccine to protect against these disorders would have a great impact on public health. A vaccine must target the M protein of group A streptococci (Streptococcus pyogenes), but until lately immunity was thought to be strain-specific and dependent on antibodies to the variable serotype-specific regions of the protein. Experiments in animals have suggested the conserved region of the M protein as a possible alternative target for protective antibodies. We constructed a 20-aminoacid peptide (peptide 145) within the conserved region of the carboxyl terminus of the protein. In mice the peptide induced serum antibodies that could opsonise reference type 5 streptococci. By enzyme-linked immunosorbent assay, positive responses to peptide 145 were obtained with serum from 77 (90%) of 86 Aboriginal subjects and 135 (81%) of 167 Thai subjects living in areas with high exposure to streptococci. Only 10 (14%) of 71 Caucasian subjects with low exposure to streptococci showed positive responses. There was no difference in the proportion positive between subjects with rheumatic heart disease and control groups (other or no heart disease). Antibodies to peptide 145 were able to opsonise isolates of streptococci from Aboriginal and Thai subjects with acute rheumatic fever as well as reference strains. This highly conserved part of the M protein may be a suitable target for vaccines to prevent streptococcal infections and their sequelae.

The emerging phenotype of long-term survivors with infantile Pompe disease

Purpose:Enzyme replacement therapy with alglucosidase alfa for infantile Pompe disease has improved survival creating new management challenges. We describe an emerging phenotype in a retrospective review of long-term survivors.Methods:Inclusion criteria included ventilator-free status and age ≤6 months at treatment initiation, and survival to age ≥5 years. Clinical outcome measures included invasive ventilator-free survival and parameters for cardiac, pulmonary, musculoskeletal, gross motor, and ambulatory status; growth; speech, hearing, and swallowing; and gastrointestinal and nutritional status.Results:Eleven of 17 patients met study criteria. All were cross-reactive immunologic material-positive, alive, and invasive ventilator-free at most recent assessment, with a median age of 8.0 years (range: 5.4–12.0 years). All had marked improvements in cardiac parameters. Commonly present were gross motor weakness, motor speech deficits, sensorineural and/or conductive hearing loss, osteopenia, gastroesophageal reflux, and dysphagia with aspiration risk. Seven of 11 patients were independently ambulatory and four required the use of assistive ambulatory devices. All long-term survivors had low or undetectable anti-alglucosidase alfa antibody titers.Conclusion:Long-term survivors exhibited sustained improvements in cardiac parameters and gross motor function. Residual muscle weakness, hearing loss, risk for arrhythmias, hypernasal speech, dysphagia with risk for aspiration, and osteopenia were commonly observed findings.Genet Med 2012:14(9):800–810.

Neonatal Thyroid Function: Effect of a Single Exposure to Iodinated Contrast Medium in Utero

PURPOSE To evaluate the effect of in utero exposure to a single dose of water-soluble intravenous iodinated contrast medium on thyroid function at birth. MATERIALS AND METHODS This study was approved by the institutional review board, with waiver of consent, and was HIPAA compliant. Maternal and newborn records were retrospectively reviewed. All pregnant women who underwent multidetector pulmonary computed tomographic angiography because they were suspected of having pulmonary embolism between 2004 and 2008 and newborns resulting from the index pregnancy were included. In all examinations, iohexol was used as the contrast agent. Dose and amount of contrast agent and gestational age at the time of administration of the contrast agent were collected, and thyroxine (T(4)) and thyroid-stimulating hormone (TSH) levels were measured at birth. A total of 344 maternal and 343 newborn records were reviewed. A descriptive analysis was performed, and means, standard deviations, and confidence intervals were reported. RESULTS Mean gestational age at the time of administration of the contrast material was 27.8 weeks +/- 7.4. The mean dose of total iodine administered was 45,000 mg/L +/- 7321. All newborns had a normal T(4) level at birth; only one newborn had a transiently abnormal TSH level at birth, which normalized at day 6 of life. This newborn was born to a mother who had many drug exposures during pregnancy. CONCLUSION A single, high-dose in utero exposure to water-soluble, low-osmolar, iodinated intravenous products, such as iohexol, is unlikely to have a clinically important effect on thyroid function at birth.

Congenital Hypothyroidism with a Delayed Thyroid-Stimulating Hormone Elevation in Very Premature Infants: Incidence and Growth and Developmental Outcomes

OBJECTIVE To test the hypothesis that very low birth weight (VLBW) and extremely low birth weight (ELBW) infants have an increased incidence of congenital hypothyroidism (CH) with a delayed thyroid-stimulating hormone (TSH) elevation and that the outcomes of these infants are similar to control infants. STUDY DESIGN Retrospective analysis of newborn thyroid screening data for 92 800 live births in Rhode Island to identify CH with a delayed TSH elevation. Developmental, growth, and endocrine outcomes of the index cases were assessed at 18 months corrected age. RESULTS CH with a delayed TSH elevation occurred in 1 in 58 ELBW, 1 in 95 VLBW, and 1 in 30 329 infants weighing ≥1500 grams (P < .0001). The incidence of head circumference <10(th) percentile was higher in VLBW infants with CH associated with a delayed TSH elevation (P < .05), and the mean head circumferences, weights, lengths, and developmental scores were similar to matched control infants. Three infants received short-term levothyroxine replacement. CONCLUSIONS The incidence of CH with a delayed TSH elevation was higher in ELBW and VLBW infants compared with infants weighing ≥1500 grams. The outcomes of these infants were comparable with matched control infants.

The role of insulin in chondrogenesis.

The ATDC5 chondrogenic cell line is typically induced to differentiate by exposure to insulin at high concentration (10 microg/ml, approximately 1600 nM). Differentiation can also be induced by physiological concentrations of insulin-like growth factor-I (IGF-I). Unlike previous reports, we observed a stimulation of differentiation, as measured by collagen X expression and Alcian Blue staining for proteoglycan synthesis, upon exposure to insulin at concentrations (10-50 nM) consistent with signaling via the insulin receptor. Analysis of lysates from proliferating and hypertrophic ATDC5 cells demonstrated that exposure to 50 nM insulin induced tyrosine phosphorylation of insulin receptors but not IGF-I receptors or hybrid receptors. In contrast to the potent effects of IGF-I to stimulate both ATDC5 proliferation and differentiation, insulin was not as potent as IGF-I as a proliferating agent but more selectively a differentiating agent. Consistent with this result, insulin was less potent than IGF-I in inducing activation of the Erk1/Erk2 mitogenic signaling pathway. Furthermore, Erk pathway inhibition did not enhance the differentiating effects of insulin as it does in the case of IGF-I exposure. Extending our observations to fetal rat metatarsal explants, we observed significant stimulation of bone growth by 50 nM insulin. This could be accounted for by a disproportionate stimulatory effect on growth of the hypertrophic zone. The proliferative zone was not significantly affected. Based on our results in both ATDC5 cells and metatarsal explants, we conclude that the insulin functioning through insulin receptor has a dominant effect as an inducer of chondrocyte differentiation. These results support assignment of a physiological role for this hormone in linear bone growth.

mTOR signaling contributes to chondrocyte differentiation

The mammalian Target Of Rapamycin (mTOR) is a nutrient‐sensing protein kinase that regulates numerous cellular processes. Fetal rat metatarsal explants were used as a physiological model to study the effect of mTOR inhibition on chondrogenesis. Insulin significantly enhanced their growth. Rapamycin significantly diminished this response to insulin through a selective effect on the hypertrophic zone. Cell proliferation (bromodeoxyuridine incorporation) was unaffected by rapamycin. Similar observations were made when rapamycin was injected to embryonic day (E) 19 fetal rats in situ. In the ATDC5 chondrogenic cell line, rapamycin inhibited proteoglycan accumulation and collagen X expression. Rapamycin decreased content of Indian Hedgehog (Ihh), a regulator of chondrocyte differentiation. Addition of Ihh to culture medium reversed the effect of rapamycin. We conclude that modulation of mTOR signaling contributes to chondrocyte differentiation, perhaps through its ability to regulate Ihh. Our findings support the hypothesis that nutrients, acting through mTOR, directly influence chondrocyte differentiation and long bone growth. Developmental Dynamics 237:702–712, 2008.

Exacerbation of the ochronosis of alkaptonuria due to renal insufficiency and improvement after renal transplantation.

In alkaptonuria, homogentisate 1,2-dioxygenase deficiency causes tissue accumulation of homogentisic acid (HGA), followed by signs and symptoms of ochronosis. These include massive urinary excretion of HGA, arthritis and joint destruction, pigmentation of cartilage and connective tissue, and cardiac valve deterioration. We describe a 46-year-old man with alkaptonuria and diabetic renal failure whose plasma HGA concentration was twice that of any other alkaptonuria patient, and whose ochronosis progressed much more rapidly than that of his two alkaptonuric siblings. After renal transplantation, the plasma HGA normalized, and the daily urinary excretion of HGA decreased by 2-3g. This case illustrates the critical role of renal tubular secretion in eliminating HGA from the body, and suggests that renal transplantation in a uremic patient not only restores HGA excretion, but may also provide homogentisate 1,2-dioxygenase activity for the metabolism of HGA.

The Effect of Rapamycin on Bone Growth in Rabbits

mTOR is a nutrient‐sensing protein kinase that regulates numerous cellular processes. Our prior studies using the mTOR inhibitor, rapamycin, indicate an important role for mTOR in chondrogenesis. We extended our observations to a physiological, in vivo model of bone growth, direct infusion of rapamycin into the proximal tibial growth plates of rabbits. Rapamycin or DMSO vehicle was infused directly into growth plates by an osmotic minipump for 8 weeks. Tibial growth was followed radiographically. At the end of the experiment, growth plates were recovered for histological analysis. Six animals were studied. No untoward effects of rapamycin infusion were found. Bone growth of limbs exposed to rapamycin was slower than control limbs, particularly during the period of most rapid growth. Histological analysis revealed that growth plate height in the rapamycin‐infused limbs was reduced. Both the hypertrophic and proliferative zones were significantly smaller in the rapamycin‐infused limbs. Direct infusion of rapamycin into proximal tibial growth plates decreased the size of the growth plate and inhibited overall long bone growth. Rapamycin appears to affect both the proliferative and hypertrophic zones of the tibial growth plate. Our results indicate that nutrients may exert a direct effect on long bone growth via mTOR‐mediated modulation of chondrogenesis at the growth plate. and suggest that the possible inhibitory effects of rapamycin on skeletal growth warrant further attention before its use in children.

The mechanism of ascorbic acid-induced differentiation of ATDC5 chondrogenic cells

The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to that observed during endochondral bone formation. Previous investigators have induced ATDC5 cells to differentiate by exposing them to insulin at high concentrations. We have observed spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing alpha-MEM. A comparison of the differentiation events in response to high-dose insulin vs. ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, Sox9, Indian hedgehog, and collagen X. We took advantage of the action of ascorbic acid to examine signaling events associated with differentiation. In contrast to high-dose insulin, which downregulates both IGF-I and insulin receptors, there were only minimal changes in the abundance of these receptors during ascorbic acid-induced differentiation. Furthermore, ascorbic acid exposure was associated with ERK activation, and ERK inhibition attenuated ascorbic acid-induced differentiation. This was in contrast to the inhibitory effect of ERK activation during IGF-I-induced differentiation. Inhibition of collagen formation with a proline analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to differentiate in the absence of ascorbic acid. Our results indicate that matrix formation early in the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation. We conclude that ascorbic acid can promote the differentiation of ATDC5 cells by promoting the formation of collagenous matrix and that matrix formation mediates activation of the ERK signaling pathway, which promotes the differentiation program.

Expansion of the clinical phenotype associated with mutations in activity-dependent neuroprotective protein

Activity-dependent neuroprotective protein (ADNP) is a highly conserved transcription factor comprised of nine-zinc finger domains and a homeobox domain.1 ,2 It is highly expressed prenatally during critical stages of embryonic brain development.3 Knockout (KO) mouse embryos demonstrate severe neurodevelopmental morphological profiles.4 Although the ADNP KO is lethal, heterozygous embryos demonstrate typical embryogenesis yet display a neurodevelopmental delay phenotype including decreased neuronal survival.3 ,5 Exome sequencing in the Simons Simplex Collection autism dataset identified ADNP mutations as a putative autism gene candidate.6 ,7 Helsmoortel et al 8 recently reported 10 individuals with autism spectrum disorder (ASD) and mutations in exon 5 of the ADNP gene, nine of which were confirmed de novo. These patients also exhibited intellectual disability (ID) and dysmorphic features such as a prominent forehead. Mutations in the ADNP gene are estimated to be present in at least 0.17% of ASD cases. The current report further expands the ADNP phenotype to include abnormalities in the developing visual system (such as eye movement abnormalities and cortical visual impairment). We advise appropriate screening of eye movement and visual symptoms by clinicians in patients who have mutations in ADNP . The 6-year-old patient was the first child born to healthy non-consanguineous parents. Pregnancy was notable for placenta previa and early dilation and effacement of the cervix 3 weeks prior to delivery. The patient was born at 40 weeks via C-section secondary to failure to progress and maternal (i.e. maternal hypertension) hypertension weighing 6 pounds 14 ounces. She had a short stay in the neonatal intensive care unit (NICU) for breathe holding and feeding problems. She was also hospitalised at 6 weeks for an acute life-threatening event of multiple cyanotic episodes thought to be due to breath holding. Our patient has been diagnosed with hypotonia and mixed developmental …