Osteogenesis imperfecta and the teeth, eyes, and ears-a study of non-skeletal phenotypes in adults.

Osteogenesis imperfecta (OI) is a disease causing bone fragility; however, it potentially affects all organs with a high content of collagen, including ears, teeth, and eyes. The study is cross-sectional and compares non-skeletal characteristics in adults with OI that clinicians should be aware of when caring for patients with OI. INTRODUCTION: Osteogenesis imperfecta (OI) is a hereditary connective tissue disorder. The skeletal fragility is pronounced; however, OI leads to a number of extra-skeletal symptoms related to the ubiquity of collagen type 1 throughout the human body. The vast majority of knowledge is derived from studies performed in the pediatric population. Thus, we aimed to investigate the nature and prevalence of ophthalmologic, odontologic, and otologic phenotypes in an adult population with OI. METHODS: The study population comprises 85 Danish OI patients (age 44.9 ± 15.9 years). Fifty-eight patients had OI type I, 12 OI type III, and 15 OI type IV according to the classification by Sillence. Audiometric evaluations and dental examinations were performed in 62 and 73 patients, respectively. Ophthalmologic investigations were performed in 64 patients, including measurements of the central corneal thickness. RESULTS: All patients, except two, had corneal thickness below the normal reference value. Patients with OI type I and patients with a quantitative collagen defect had thinner corneas compared to patients with OI type III and other patients with a qualitative collagen defect. One patient in this cohort was diagnosed with and treated for acute glaucoma. Dentinogenesis imperfecta was diagnosed in one fourth of the patients, based on clinical and radiographic findings. This condition was predominately seen in patients with moderate to severe OI. Hearing loss requiring treatment was found in 15 of 62 patients, of whom three were untreated. The most prevalent type of hearing loss (HL) was sensorineural hearing loss, whereas conductive HL was solely seen in patients with OI type III. The patients with the most severe degrees of HL were patients with mild forms of OI. Age was associated with increased HL. CONCLUSION: Although significant health problems outside the skeleton are frequent in adult patients with OI, the patients are not consistently monitored and treated for their symptoms. Clinicians treating adult patients with OI should be aware of non-skeletal health issues and consider including regular interdisciplinary check-ups in the management plan for adult OI patients.

A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes.

The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 microM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.

The effect of low temperature and fasting during the winter on metabolic stores and endocrine physiology (insulin, insulin-like growth factor-I, and thyroxine) of coho salmon, Oncorhynchus kisutch.

The objective of this study was to examine the effect of winter feeding and fasting at both high (10 degrees ) and low (2.5 degrees ) temperatures on growth, metabolic stores, and endocrinology of coho salmon. Treatments were as follows: warm-fed, warm-not fed, cold-fed, and cold-not fed during the winter (January-February). The following parameters were measured: length, weight, whole body lipid, liver glycogen, hepatosomatic index, and plasma levels of insulin, insulin-like growth factor-I (IGF-I), and thyroxine (T4). Warm-fed fish grew continuously throughout the experiment from 21.5 +/- 0.3 to 43.4 +/- 1.4 g and were larger than fish in the other treatments. Fish in all other treatments grew from 21.5 +/- 0.3 to approximately 32 g and showed depressed growth during January and February. During the winter, liver glycogen, hepatosomatic index, plasma insulin, and IGF-I were highly influenced by manipulations in rearing conditions, whereas whole body lipid and plasma T4 were less affected. Plasma insulin levels fluctuated dramatically (from 2 to 7 ng/ml) in the two cold-acclimated groups shortly after the change in temperature. In general, the plasma insulin levels of the warm-fed fish were the highest (8-9 ng/ml), those of the warm-not fed fish were the lowest (2-5 ng/ml), and those of the two cold-acclimated groups were more variable but intermediate. In contrast, plasma IGF-I levels showed a decline with temperature decrease (from 9 to 5 ng/ml) and more gradual changes than insulin with the change in feeding. The highest plasma IGF-I levels were found in the warm-fed fish (10-15 ng/ml), the lowest levels were in the cold-not fed fish (4-5 ng/ml), and those of the warm-not fed and cold-fed fish were intermediate. During the treatment period the T4 levels were relatively unaffected by manipulations in feeding and temperature compared with either insulin or IGF-I. These data suggest that the insulin, IGF-I, and thyroid axes are differentially regulated under changing seasonal and/or environmental conditions in yearling salmon.

Effects of ration on somatotropic hormones and growth in coho salmon.

We examined the response of growth hormone (GH), total plasma insulin-like growth-factor I (IGF-I), and growth rate to a change in ration in coho salmon. Tanks of individually tagged fish were placed on high, medium, or low ration, and sampled every 2 weeks for 8 weeks to create a range of growth rates. Some fish received non-lethal blood draws, while others were sampled terminally. Plasma IGF-I levels were higher in high ration fish than in low ration fish from 4 weeks after the beginning of experimental diets to the end of the experiment. GH levels were low and similar in all fish after changing rations, except for the fish in the low ration group at week 2. IGF-I was strongly correlated with specific growth rate in weight in terminally sampled fish after 4 weeks. GH did not correlate with growth rate or IGF-I levels. Growth parameters (length, weight, specific growth rates in weight and length, and condition factor) responded to ration. Serial sampling reduced growth rates and hematocrit, but did not change hormone levels. This study shows that IGF-I responds to changed rations within 2-4 weeks in salmonids.

Long-term peripheral treatment of immature coho salmon (Oncorhynchus kisutch) with human leptin has no clear physiologic effect.

The effects of long-term peripheral exposure to recombinant human leptin were tested in immature coho salmon under both fed and fasted conditions. We found that high circulating levels of human leptin did not alter growth, energy stores, gonad weight, pituitary content of follicle-stimulating hormone, or plasma levels of insulin-like growth factor-I, insulin, growth hormone, or thyroxine.

Insulin-like growth factor-I and environmental modulation of growth during smoltification of spring chinook salmon (Oncorhynchus tshawystscha).

The relations among rearing environment, fish size, insulin-like growth factor-I, and smoltification were examined in yearling spring chinook salmon (Oncorhynchus tshawytscha). Juvenile chinook salmon were size-graded into small and large categories. Half of the fish in each group were reared at an increased temperature and feeding rate beginning in mid-February, resulting in four distinct treatment groups: large warm-water (LW), large cool-water (LC), small warm-water (SW), and small cool-water (SC). Increased temperature and feeding rate resulted in overall higher growth rates for the LW and SW groups. Temporal increases in insulin-like growth factor-I (IGF-I) were found in all groups through the spring. Plasma IGF-I levels were significantly higher in warm-water groups than in cool-water groups from late March through May. Size itself appeared to have little relation to plasma IGF-I levels. Simple regression showed a significant relation between plasma IGF-I and growth (P < 0. 001, R2 = 0.50). No differences were found between treatment groups in other physiological parameters assessed (plasma thyroxine, gill Na+-K+-ATPase, liver glycogen, body lipid).

In vitro thyrotropin-releasing activity of corticotropin-releasing hormone-family peptides in coho salmon, Oncorhynchus kisutch.

Investigations of hypothalamic regulation of fish thyrotropin (TSH) secretion and subsequent thyroid activity have been impeded by the lack of a reliable assay for TSH. Using a recently developed radioimmunoassay (RIA) for coho salmon TSH we employed an in vitro pituitary cell culture technique to examine regulation of TSH secretion by corticotropin-releasing hormone (CRH) family peptides [ovine CRH (oCRH), carp urotensin I (UI), and frog sauvagine (SV)] as well as thyrotropin-releasing hormone (TRH), salmon growth hormone-releasing hormone (sGHRH), and salmon gonadotropin-releasing hormone (sGnRH). At concentrations of 0.01 to 100 nM, TRH, sGHRH, and sGnRH did not stimulate TSH secretion from coho salmon pituitary cells. However, at these same concentrations, both oCRH and SV caused a significant and concentration-dependent increase in TSH secretion; whereas, UI was highly stimulatory at all concentrations tested. In a related experiment we examined the effect of alpha-helical CRF(9-41) on oCRH-stimulated TSH release by pituitary cells. alpha-Helical CRF(9-41) is an analogue of CRH that has been shown by others to antagonize the adrenocorticotropic hormone (ACTH)-releasing activity of CRH in goldfish. Preincubation of cells with 1 microM alpha-helical CRF(9-41) for 4 h caused a significant suppression of the TSH-releasing activity of oCRH at 1.0 and 10 nM concentrations. The results of these experiments demonstrate the potency of a CRH-like peptide in the hypothalamic regulation of TSH in fish and reveal similarities in the inhibition of the response of both the thyroid and interrenal axis of fish to alpha-helical CRF(9-41).

Effects of gonadectomy on plasma gonadotropins I and II in coho salmon, Oncorhynchus kisutch.

Studies of both salmon and trout have indicated that the levels of gonadotropins, GTH I and GTH II, in the pituitary and peripheral circulation vary during the reproductive cycle. To evaluate the possible feedback relationship between the gonads and pituitary GTH secretion, we studied the effects of bilateral gonadectomy on plasma levels of GTH I and GTH II in coho salmon, Oncorhynchus kisutch. During late spermatogenesis in males and late vitellogenesis in females, plasma GTH I levels increased significantly after gonadectomy, approximately 6- and 5-fold over presurgery levels at 3 and 14 days after surgery, respectively, and then declined to near presurgery levels by Day 17. No change in GTH I levels occurred in sham-operated fish. In all groups, GTH II levels were nondetectable and did not change significantly up to 17 days postsurgery. In males gonadectomized during spermiation, plasma GTH I levels increased significantly, approximately 10-fold over presurgery levels by 7 days postsurgery, and remained elevated thereafter. In contrast to the males in late spermatogenesis, the spermiating fish had detectable levels of GTH II (2-3 ng/ml), and significant elevations in plasma GTH II levels (approximately 60-fold) were observed 7 days after gonadectomy. These experiments demonstrate that the gonads exert negative feedback effects on secretion of both GTH I and GTH II, but the effect varies seasonally and the nature of the specific factor(s) from the gonads that inhibits and/or stimulates GTH production and secretion remains to be clarified.

Quantification of salmon alpha- and thyrotropin (TSH) beta-subunit messenger RNA by an RNase protection assay: regulation by thyroid hormones.

In order to study salmon thyroid-stimulating hormone (TSH), we designed a highly specific, sensitive, and rapid RNase protection assay (RPA) for quantification of steady-state levels of salmon TSH beta-subunit mRNA expression. The cDNA encoding the beta-subunit of TSH was isolated from coho salmon pituitary total RNA by reverse-transcriptase PCR, partially sequenced, and used as template for synthesizing a radioactively labeled, sequence-specific, antisense probe, and sense standard for the RPA. This assay, along with a similar RPA previously designed for coho salmon total alpha-subunit mRNA, was used to examine the effects of feeding T3 (0, 10, 100 micrograms/g) and methimazole (a thyroid inhibitor) (2.5 mg/g) on TSH subunit gene expression after 2 and 4 weeks. The low dose of T3 (10 micrograms/g) caused no change in TSH beta mRNA after 2 and 4 weeks and a transient increase in alpha mRNA after 2 weeks, followed by no significant effect after 4 weeks. The high dose of T3 (100 micrograms/g) caused a decrease in TSH beta mRNA after 4 weeks and no change in total alpha mRNA after 2 and 4 weeks. In contrast, methimazole treatment caused significant increases in both TSH beta mRNA (250%) and alpha mRNA (50%) levels after 4 weeks. These findings confirm that, as in mammals, TSH alpha- and beta-subunit expression in teleosts may be differentially regulated by negative feedback from the thyroid hormones.

Salmon thyroid-stimulating hormone: isolation, characterization, and development of a radioimmunoassay.

Coho salmon (Oncorhynchus kisutch) thyroid-stimulating hormone (TSH) was isolated by ethanol extraction of pituitary glands from mature coho salmon. Extraction was followed by gel-filtration chromatography on Sephadex G-100 superfine, anion-exchange chromatography on a Whatman DE-52 column, and finally by reverse-phase high-performance liquid chromatography. Fractions were monitored for TSH content by a homologous in vivo bioassay and by immunoblots using anti-human TSH beta-subunit antisera. In vivo treatment of coho salmon parr with coho salmon TSH caused a dose-dependent increase in plasma thyroxine level similar to that induced by bovine TSH. The N-terminal sequence (25 residues) of the salmon TSH beta subunit has 56% sequence identity to that of human TSH beta subunit and is identical to the deduced amino acid sequence of trout TSH beta subunit. The N-terminal sequence (25 residues) of the salmon TSH alpha subunit is identical to gonadotropin alpha-II subunit. Molecular sizes of the alpha and beta subunits are 18,000 and 24,000 daltons, respectively, as estimated by SDS-PAGE. Antiserum generated against salmon TSH, which was preadsorbed with alpha subunit using an alpha-subunit affinity column, detected only salmon TSH beta subunit by immunoblot and specifically stained thyrotropin-producing cells of the pituitary gland. A homologous radioimmunoassay (RIA) was developed using purified salmon TSH standard, iodinated TSH beta subunit, and antiserum generated against salmon TSH. Cross-reactivities of GTH I, GTH II, GTH I beta and GTH II beta subunits, alpha subunit, growth hormone, prolactin, and somatolactin were less than 1%. Displacement curves for serial dilutions of plasma and pituitary extracts of various salmonid species, as well as coho salmon pituitary cell culture medium, were parallel to the coho salmon TSH standards. In contrast, plasma of hypophysectomized juvenile coho salmon and pituitary extracts of Pacific tomcod (Microgadus proximus) did not displace bound radiolabeled salmon TSH. Finally, in vivo injection of juvenile coho salmon with triiodothyronine decreased plasma TSH levels, whereas the goitrogen, methimazole, increased plasma TSH levels. Injection of gonadotropin-releasing hormone agonist did not alter plasma TSH. These data suggest that the RIA is specific for TSH and confirm a negative-feedback relationship between the thyroid hormones and TSH.

A new approach to alcoholism detection in primary care.

This article describes a model that brings together the chemical dependency, mental health, and primary care services of a staff model HMO for the purpose of establishing a primary care clinic-based program to assist physicians in early detection of chemical dependency and frequent psychiatric disorders. The model creates a partnership between a master's-level professional social worker (MSW) and a designated family physician from the clinic. Their focus is on provider education, consultation, and on assisting patients with referrals to the appropriate services. Parameters of success include changes on referral patterns, use of the MSW's services, and clinic satisfaction. In addition, there are indications that early intervention has had a positive impact on subsequent use of other health care system's resources.