Clinical and radiologic factors associated with pulmonary nodule etiology in organ transplant recipients.

Identifying clinical and radiographic factors that are associated with a specific etiology of pulmonary nodules (PNs) in solid-organ transplant (SOT) recipients might be helpful in guiding empiric therapy. Multivariable logistic regression was used to assess the relationship of clinical and radiographic variables to the etiology of PN in a retrospectively identified cohort of SOT recipients at a single transplant center. PNs in 55 SOT recipients (lung 15%, heart 22%, liver 42%, kidney 18% or kidney/pancreas 5%) were diagnosed at a mean of 1061 days post-transplant and were infectious in 31 of 55 (56%) (bacterial 22%, fungal 33%, viral 2%) and noninfectious in 24 of 55 (44%) [post-transplant lymphoproliferative disorder (PTLD) 25%, carcinoma 18%]. Radiographic 'consolidation' was independently associated with an infectious etiology (OR, 20.2, p < 0.01). Epstein-Barr virus seronegativity and lung transplant were each associated with PTLD (OR, 21.7, p < 0.01) and (OR, 36.6, p < 0.001), respectively. Diagnosis less than 90 days post-transplant was associated with Aspergillus infection (OR, 12.9, p = 0.007). Specific clinical and radiographic features are associated with specific etiologies of PNs in SOT recipients and might be useful for guiding empiric therapy while awaiting results of definitive diagnostic studies.

Calcium is an equipotent stimulator of stanniocalcin secretion in freshwater and seawater salmon.

Stanniocalcin (STC) is a calcium- and phosphate-regulating hormone produced by the corpuscles of Stannius in fishes. A rise in ion calcium (Ca2+) levels is the principal stimulus for secretion, and the hormone acts on the gills, gut, and kidneys to restore normocalcemia. The STC-producing cells in marine fishes are metabolically more active and secrete more hormone than those in freshwater fishes, which has been attributed to the higher calcium content of seawater placing a greater burden on the organ systems governing Ca2+ homeostasis. In this study we have addressed the question of whether or not the STC cells in marine fishes are more sensitive to Ca2+, by comparing the secretagogic effects of Ca2+ in freshwater- and seawater-adapted coho salmon. The results showed that the STC cells were equally Ca(2+)-sensitive in the two groups. Therefore, in spite of the fact that the STC cells are more active in marine fishes this requires no apparent adjustment in cellular sensitivity to calcium.

Immunological and biological evidence for a stanniocalcin-like hormone in human kidney.

The corpuscles of Stannius are responsible for the synthesis and secretion of stanniocalcin (STC), a glycoprotein hormone that regulates calcium and phosphate homeostasis in fishes through its actions on the gills and kidneys. The corpuscles of Stannius and STC are considered to be an endocrine system that is unique to fishes. In this report, we provide evidence for the existence of STC-like proteins in vertebrates other than fishes, in particular, humans. By using a well-characterized RIA for salmon STC, sera from vertebrates as diverse as sharks and humans contained measurable levels of STC-like immunoreactivity in the concentration range commonly observed in fishes, and all of these sera exhibited parallelism in the assay. By using Western blot analysis, proteins were also identified in human kidney extracts that shared several properties with the fish hormone in addition to their cross-reactivity with salmon STC antiserum. The same antiserum was used to identify a discrete population of cells in human kidney tubules that could be the source of serum immunoreactivity. Human kidney extracts containing the STC-immunoreactive proteins also had STC-related effects when injected into fishes. Collectively, the data suggest that STC may be more widespread among the vertebrates than is currently accepted.

Calcitonin: discovery, development, and clinical application.

In 1954, when I gave a talk on calcium homeostasis at the first Gordon Conference on Bones and Teeth, it was recognized that the level of ionic calcium in the plasma and body fluids must be maintained with precision, since it is critically important for a number of vital processes. However, very little was known of the mechanisms involved and I decided to make this the focus of my research career. With the assistance of a number of first-year medical students working during the summer, we developed a precise method for measuring calcium, demonstrated the remarkable constancy of plasma calcium in normal human subjects, and found that normal calcium levels were restored quickly after being artificially raised or lowered. We focussed on parathyroid hormone (PTH), which plays a key role in controlling hypocalcemia by stimulating osteolysis. While studying the control of its secretion in 1961, we discovered a second calcium-regulating hormone (calcitonin) which was released by hypercalcemia and lowered plasma calcium by inhibiting osteolysis. It is a straight-chain peptide with 32 amino acids and a 7-membered disulfide ring at the N terminal. It is produced by C cells which arise from the neural crest and is considered a neuropeptide hormone. It is produced in the thyroid of mammals and the ultimobranchial glands of lower vertebrates. We were involved in the isolation of salmon calcitonin, which is the form most widely used in therapy because of its high potency. In addition to inhibiting bone resorption, it is a powerful analgesic agent with a potency in certain circumstances which is 30-50 times that of morphine. It is widely used clinically for the treatment of Paget's disease, hypercalcemia, osteoporosis, and relief of bone pain. World sales in 1992 exceeded US$900 million, of which 85% was for osteoporosis.

The gill calcium transport cycle in rainbow trout is correlated with plasma levels of bioactive, not immunoreactive, stanniocalcin.

Stanniocalcin (STC) is an inhibitor of gill Ca2+ transport that is produced by the corpuscles of Stannius, endocrine glands in bony fish. In young rainbow trout (Oncorhynchus mykiss), there are cyclical changes in the rate of gill Ca2+ transport, with alternating phases of accelerated and reduced uptake every 14 days. Previous studies by our laboratory have established that the responsiveness of young trout to the inhibitory effects of exogenous STC is dependent on this cycle. Trout are highly responsive to STC at peaks of Ca2+ uptake and unresponsive at nadirs, which has led us to suggest that the gill Ca2+ transport cycle may be regulated by a reciprocal cycle in the levels of plasma STC. In this report, we have further characterized the gill Ca2+ transport cycle in salmonids and investigated the role of STC in its regulation. Our results showed that the cycle is synchronous and is likely a characteristic feature in all salmonids but that it varies in amplitude between species. Surprisingly, we observed no correlation between circulating levels of radioimmunoassayable STC and the rate of gill Ca2+ transport in trout. To address this apparent contradiction, trout fry were passively immunized with STC antiserum to determine if there were variable amounts of bioactive STC in the circulation, at times when trout were either more or less sensitive to exogenous STC. We observed that during the times when trout were responsive to STC treatment (i.e., cycle peaks), passive immunization had no effect on the rate of gill Ca2+ transport in fish from the same population, indicating that there were low levels of bioactive STC in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and cDNA sequence analysis of coho salmon stanniocalcin.

Stanniocalcin (STC) (formerly known as both teleocalcin and hypocalcin) is an anti-hypercalcemic, glycoprotein hormone that is produced by the corpuscles of Stannius (CS), endocrine glands that are confined to bony fishes. The hormone has a unique amino acid sequence and exists as a disulfide-linked homodimer in the native state. In previous studies, we have described the purification and characterization of two salmon STCs, and examined the regulation of hormone secretion in response to calcium using both in vitro and in vivo model systems. This report describes the molecular cloning and cDNA sequence analysis of a coho salmon STC messenger RNA (mRNA) from a salmon CS lambda gt10 cDNA library. The STC mRNA in salmon is approximately 2 kilobases in length and encodes a primary translation product of 256 amino acids. The first 33 residues comprise the prepro region of the hormone, whereas the remaining 223 residues make up the mature form of the hormone. One N-linked, glycosylation consensus sequence was identified in the protein coding region as well as an odd number of half cysteine residues, the latter of which would allow for interchain bonding or dimerization of monomeric subunits. In addition, three sites were identified in the mature protein core of STC (two dibasic, one tribasic) that may be acted upon by endopeptidases to produce truncated forms of the hormone. In support of this latter possibility, Western blot analysis revealed multiple molecular weight forms of sTC within salmon glands.

Salmon stanniocalcin and bovine parathyroid hormone have dissimilar actions on mammalian bone.

Stanniocalcin (STC), a calcium-regulating glycoprotein hormone isolated from the corpuscles of Stannius of salmon, was tested for effects on bone and calcium metabolism in mammalian species (rats and mice). STC generally failed to alter serum calcium of parathyroidectomized rats at concentrations equimolar with effective concentrations of parathyroid hormone (PTH). STC did not increase cAMP in ROS 17/2.8 or UMR-108 osteosarcoma cells, OK kidney cells, fetal rat limb bones, or neonatal mouse calvariae, and similarly failed to increase urinary cAMP in rats. STC did not consistently stimulate resorption in any of the rodent bone culture systems, although variable resorptive responses were elicited in fetal mouse calvariae. The results indicate that this fish hormone has limited, if any, PTH-like activity on calcium metabolism in mammalian systems.

Studies on the regulation and characterization of plasma stanniocalcin in rainbow trout.

Stanniocalcin (STC) is a hormone that is synthesized and secreted by the corpuscles of stannius (CS), endocrine glands that are unique to the bony fishes. The hormone inhibits Ca2+ transport from the aquatic environment into the bloodstream by way of the gills. Previous in vitro studies by our laboratory have shown that STC secretion is positively regulated by Ca2+ in a time- and dose-dependent fashion. In this report, we have examined circulating levels of STC in free-swimming, cannulated rainbow trout and how hormone levels are affected by surgical stress and intra-arterial infusions of mono and divalent cations. In addition, the plasma hormone has been concentrated by immunoaffinity chromatography and characterized by Western blot analysis. The results suggest that the in vivo response of the CS is extremely rapid and Ca(2+)-specific and that STC circulates in multiple molecular weight forms.

Effects of synthetic peptide fragments of teleocalcin (hypocalcin) on calcium uptake in juvenile rainbow trout (Salmo gairdneri).

A rainbow trout fry bioassay based on 45Ca uptake was used to compare the effects of pure coho salmon teleocalcin (TC) and several synthetic peptide fragments of TC. Calcium uptake in the fry exhibited a cycle, with an amplitude variation of 3.3 to 48.8 mumol.kg-1.hr-1 and a periodicity of 8 to 21 days. The N-terminal 1-20 amino acid peptides of both eel and salmon TC significantly inhibited 45Ca uptake at the high point of the calcium uptake cycle (up to 75%), although the effective doses of the peptides on a molar basis were 20 to 200 times that of the intact molecule. In contrast, the C-terminal fragment of eel TC (amino acids 202-231) did not have an inhibitory effect on calcium uptake. Instead, it significantly enhanced 45Ca uptake in trout fry (up to sixfold) at the low point of the calcium uptake cycle.

Processing and bioactivity of the corpuscles of Stannius protein of the Australian eel.

The primary structure of the major protein from the Corpuscles of Stannius (CS) of the Australian eel was elucidated from the cDNA sequence and was found to bear close similarity to the N-terminal amino acid sequence of the presumably homologous salmon hormone, teleocalcin (TC). The cDNA sequence predicted a preproprotein of 263 amino acids. Following removal of a 17 amino acid signal peptide, specific monobasic cleavage at an Arg-Phe bond generates the 231 amino acid mature form of the protein. The isolation and sequence determination of the prosequence confirms that the precursor contains a prosegment of 15 residues. Various fragments of the protein have been synthesized chemically and their biological activity assessed. The N-terminal 1-20 fragment of the mature protein inhibits calcium uptake in fingerling trout, the effect being similar, but not equipotent to salmon teleocalcin. Further, infusion of either the N-terminal 1-20 or the 81-94 fragment at 50 µg/h into the renal artery of conscious sheep, caused significant decreases in systemic plasma potassium concentration and in potassium excretion. The 1-20 fragment also gave rise to a small but significant increase in sodium excretion. Infusion of TC at the same rate results in a significant decrease in plasma potassium and phosphate concentration as well as a significant decrease in potassium excretion. Bovine PTH (1-34) at 100 µg/h causes a small decrease in plasma potassium and phosphate and an increase in plasma calcium concentration, and was the only peptide to cause a significant decrease in calcium excretion.

Comparative biochemistry and physiology of teleocalcin from sockeye and coho salmon.

This is a comparative study of the glycoprotein hormone, teleocalcin, from the corpuscles of Stannius of sockeye (Oncorhynchus nerka) and coho (O. kisutch) salmon. Coho teleocalcin was purified by the same procedures used previously to obtain sockeye teleocalcin and was obtained in a comparable yield. Both salmon teleocalcins had the same molecular weight as estimated by sodium dodecyl sulfate-electrophoresis and both appeared to have the structure of disulfide-linked oligomers. The two hormones were similar on the basis of amino acid and carbohydrate composition and shared 95% homology in the first 40 residues on the N-terminal. The salmon teleocalcins also shared 80% homology with the predicted 1-40 N-terminal sequence from Australian eels (Anguilla australis). Both teleocalcins had potent inhibitory effects on gill calcium uptake in intact rainbow trout (Salmo gairdneri). However, these effects were observed only at the peak in the calcium uptake cycle that is displayed by this species. In North American eels (A. rostrata), the acute administration of both teleocalcins caused significant inhibition of gill calcium uptake without any concomitant changes in plasma calcium levels or other plasma electrolytes. In 4- and 7-day stanniectomy (STX) eels, the acute administration of coho teleocalcin significantly reduced or completely abolished the accelerated gill calcium transport that occurs postoperatively, with no concomitant changes in plasma electrolytes or post-STX hypercalcemia. These experiments provide further evidence that teleocalcin is a regulator of gill calcium transport and has no acute hypocalcemic effects in fish.

Development of a primary culture system for rainbow trout corpuscles of stannius and characterization of secreted teleocalcin.

A primary culture system has been established for rainbow trout corpuscles of Stannius (CS). A RIA has also been developed to monitor and characterize the teleocalcin secreted by these cultures. The primary cultured CS cells actively synthesized and secreted teleocalcin for up to 39 days when maintained in amphibian and mammalian culture media. Numerous teleocalcin cells were also evident after immunocytochemical staining of the CS cultures. When the cultures were labeled with L-[35S]methionine, de novo synthesized and secreted teleocalcin was judged to be a glycosylated protein on the basis of Concanavalin-A-Sepharose binding and was similar in size to the intracellular form of the hormone. This culture system may prove to be ideal for identifying those factors that regulate teleocalcin secretion.

Immunological studies on teleocalcin and salmon corpuscles of Stannius.

This report describes the characterization of an antiserum to salmon teleocalcin, a glycoprotein hormone that has recently been isolated from sockeye salmon, Oncorhynchus nerka, corpuscles of Stannius (CS). Immunodiffusion studies showed that teleocalcin was immunologically identical in four species of Pacific salmon. Histological staining of adjacent sections of sockeye salmon CS by periodic acid-Schiff reagent and immunocytochemistry revealed that the teleocalcin-immunoreactive cells were also periodic acid-Schiff positive. In bioassays using rainbow trout (Salmo gairdneri), the biological effects of teleocalcin were completely abolished by pretreatment with the antiserum before injection. The antiserum was ineffective by itself, however, in neutralizing endogenous teleocalcin. Immunoprecipitation studies of in vitro translation products from salmon CS poly(A)+ RNA identified a 26K protein, which corresponds to the size of the teleocalcin monomer as predicted by cDNA sequencing studies. Similar banding patterns were obtained with purified teleocalcin and crude CS extracts by Western blot analysis. However, there was one extra band (32K) in the CS extracts that was not present in the purified hormone preparations. This may represent the pro-form of the hormone in salmon.

Chromogranin A is present in and released by fish endocrine tissue.

Chromogranin A (CgA) is a protein that is present in many mammalian endocrine cells and co-secreted with their resident hormones. We have demonstrated the presence of CgA by immunohistology in the ultimobranchial glands and corpuscles of Stannius of rainbow trout. CgA was also detected by radioimmunoassay in the medium of incubated coho salmon ultimobranchial glands. Our observations demonstrate the presence of CgA in endocrine glands of evolutionarily divergent species. These observations are consistent with the hypothesis that CgA participates in the secretory process of a wide variety of hormones.

Purification, characterization, and bioassay of teleocalcin, a glycoprotein from salmon corpuscles of Stannius.

This report describes the isolation of teleocalcin, a glycoprotein hormone from the corpuscles of Stannius (CS) of sockeye salmon (Oncorhynchus nerka), using affinity, gel exclusion, and ion exchange chromatography. In discontinuous, gradient (10-20%) SDS gels under nonreducing conditions, teleocalcin had an estimated molecular weight of of 39,300 and migrated as a single band. Two bands with estimated molecular weights of 28,000 and 32,000 were obtained upon reduction and carboxymethylation of the molecule. In acid-urea gels, teleocalcin migrated as one band with an Rf of 0.17. Amino acid sequence analysis revealed single residues for the first 19 amino acids, with phenylalanine as the N-terminal residue. Teleocalcin did not resemble parathyroid hormone (PTH) with respect to amino acid composition and exhibited no cross-reactivity in two PTH radioimmunoassays. Gas-liquid chromatographic analysis of teleocalcin demonstrated that mannose was the principal sugar present (1.86%) and glucosamine was the only hexosamine identified (2.49%). Smaller quantities of galactose (0.49%), fucose (0.44%), and sialic acid (1.6%) were also found. In bioassays using juvenile rainbow trout (Salmo gairdneri), teleocalcin significantly reduced the rate of net branchial 45Ca uptake at dosages as low as 0.02 microgram/g body wt. This inhibitory effect was dependent upon the branchial calcium uptake cycle that has been identified in this species. Pronounced inhibition was observed during periods of high uptake whereas little or no effect was obtained during the low uptake phase. The results are discussed in relation to a possible role of teleocalcin in regulating this cycle.