Diffusion MRI correlation with p16 status and prediction for tumor progression in locally advanced head and neck cancer.

Purpose: To investigate p16 effects on diffusion image metrics and associations with tumor progression in patients with locally advanced head and neck cancers. Methods: Diffusion images pretreatment and after 20 Gy (2wk) of RT were analyzed in patients with cT4/N3 p16+ oropharynx cancer (OPSCC) (N=51) and locoregionally advanced head and neck squamous cell carcinoma (LAHNSCC) (N=28), enrolled onto a prospective adaptive RT trial. Mean ADC values, subvolumes with ADC <1.2 um2/ms (TVLADC), and peak values of low (µL) and high (µH) components of ADC histograms in primary and total nodal gross tumor volumes were analyzed for prediction of freedom from local, distant, or any progression (FFLP, FFDP or FFLRDP) using multivariate Cox proportional-hazards model with clinical factors. P value with false discovery control <0.05 was considered as significant. Results: With a mean follow up of 36 months, 18 of LAHNSCC patients and 16 of p16+ OPSCC patients had progression. After adjusting for p16, small µL and ADC values, and large TVLADC of primary tumors pre-RT were significantly associated with superior FFLRDP, FFLP and FFDP in the LAHNSCC (p<0.05), but no diffusion metrics were significant in p16+ oropharynx cancers. Post ad hoc analysis of the p16+ OPSCC only showed that large TVLADC of the total nodal burden pre-RT was significantly associated with inferior FFDP (p=0.05). Conclusion: ADC metrics were associated with different progression patterns in the LAHNSCC and p16+ OPSCC, possibly explained by differences in cancer biology and morphology. A deep understanding of ADC metrics is warranted to establish imaging biomarkers for adaptive RT in HNSCC.

Differential Alphavirus Defective RNA Diversity between Intracellular and Extracellular Compartments Is Driven by Subgenomic Recombination Events.

Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective viral RNAs featuring large deletions during replication. These defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity-of-infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early-passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated next-generation sequencing libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than P2 virions and that specific populations of D-RNAs are differentially expressed between intracellular and extracellular compartments. Importantly, these trends were likewise observed in a murine model of CHIKV AF15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that is conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which were confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.IMPORTANCE Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between the capsid-E3 region and the E1-3' untranslated region (UTR) and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally, they may exist as subgenome-derived transcripts.

Development of a human stanniocalcin radioimmunoassay: serum and tissue hormone levels and pharmacokinetics in the rat.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fish and recently identified in humans and other mammals. In fish STC is produced by one gland, circulates freely in the blood and plays an integral role in mineral homeostasis. In mammals, STC is produced in a number of different tissues and serves a variety of different functions. In kidney, STC regulates phosphate reabsorption by proximal tubule cells, whereas in ovary it appears to be involved in steroid hormone synthesis. However there is no information on circulating levels of STC in mammals or the regulation of its secretion. In this report we have developed a radioimmunoassay (RIA) for human STC. The RIA was validated for measuring tissue hormone levels. However human and other mammalian sera were completely devoid of immunoreactive STC (irSTC). To explore the possibility that mammalian STC might have a short half-life pharmacokinetic analysis was carried out in rats. STC pharmacokinetics were best described by a two compartment model where the distribution phase (t1/2(alpha)) equaled 1 min and the elimination phase (t1/2(beta)) was 60 min. However the STC in the elimination phase no longer crossreacted in the RIA indicating it had undergone substantial chemical modification, which could explain our inability to detect irSTC in mammalian sera. When we compared the pharmacokinetics of human and fish STC in mammalian and fish models the human hormone was always eliminated faster, indicating that human STC has unique structural properties. There also appears to be a unique clearance mechanism for STC in mammals. Hence there are major differences in the delivery and biology of mammalian STC. Unlike fishes, mammalian STC does not normally circulate in the blood and functions instead as a local mediator of cell function. Future studies will no doubt show that this has had important ramifications on function as well.

Stanniocalcin in the seawater salmon: structure, function, and regulation.

Stanniocalcin (STC) is a homodimeric glycoprotein hormone that was first discovered in fish, where it is produced by unique endocrine glands known as the corpuscles of Stannius (CS). In freshwater salmon, STC plays an integral role in Ca2+ and phosphate homeostasis. High levels of extracellular Ca2+ promote the synthesis and release of STC, which on entering the bloodstream reduces the levels of gill and gut Ca2+ transport and renal phosphate excretion to restore normocalcemia. In this report, we have examined STC in seawater salmon. We have studied the distribution of STC protein and mRNA in marine Atlantic salmon CS cells, the responsiveness of these cells to Ca2+, and some physical properties of the hormone. Our results demonstrated that all Atlantic salmon CS cells expressed the STC gene. Furthermore, these cells exhibited a Ca2+ sensitivity that was remarkably similar to those in freshwater salmon in terms of its ability to stimulate STC secretion and gene expression. When Atlantic salmon glands were fractionated by concanavalin A (ConA)-Sepharose chromatography, two distinct forms of the hormone were identified, both of which were recognized by sockeye salmon STC antiserum, and designated as STC1 and STC2. STC1 was a glycosylated, 42-kDa disulfide-linked dimer, with a high affinity for ConA. STC2 did not bind to ConA, was 44 kDa in size, and had a different subunit structure. STC2 was also a less effective inhibitor of gill Ca2+ transport in fish. Collectively, the results suggest that there is a second form of STC in salmon.

Development of a dose-response bioassay for stanniocalcin in fish.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fish and recently identified in mammals. In fish, STC is released into the bloodstream in classical endocrine fashion and has well established regulatory effects on calcium and phosphate homeostasis. However, there are no suitable dose-response bioassays for STC and consequently no methods for assigning units of potency to preparations of the hormone. All the available in vitro bioassays are too complex from a technical standpoint to readily accommodate the large number of samples required in dose-response bioassays. Most in vivo bioassays are hampered by the fact that fish have natural rhythms governing plasma STC levels which tend to make them variably sensitive to the injected hormone. In this report we have developed a new in vivo bioassay for STC using rainbow trout. The key feature of the bioassay involves suppressing plasma STC levels to the extent that fish are always receptive to injected hormone. This has been accomplished by phosphate-loading the animals, which lowers their plasma calcium levels, removes the stimulus for STC secretion and brings about a reduction in resting plasma hormone levels. The net effect is an animal that is always responsive to injected STC. With this bioassay we have been able to obtain sensitive and reproducible, dose-related effects of salmon STC on gill calcium transport.

Human stanniocalcin inhibits renal phosphate excretion in the rat.

Stanniocalcin (STC) is a glycoprotein hormone first identified in bony fishes where it counteracts hypercalcemia by inhibiting gill calcium uptake and stimulating renal inorganic phosphate (Pi) reabsorption. Human STC (hSTC) has recently been cloned and sequenced and is highly homologous to the fish hormone at the amino acid level. The objective of this study was to examine the possible effects of hSTC on electrolyte homeostasis and renal function in the rat. Recombinant hSTC was expressed in bacteria and purified by metal-ion affinity chromatography and reverse-phase high performance liquid chromatography. Anesthetized animals were given bolus infusions of 1, 5, or 10 nmol hSTC per kilogram of body weight. Control animals received solvent alone. The most effective dosage was 5 nmol/kg, which caused significant reductions in both absolute and fractional phosphate excretion in comparison with control rats. The hSTC had no effect on the renal excretion of other ions, the glomerular filtration rate, renal blood flow, blood pressure, or plasma electrolytes (Na+, K+, Ca2+, Pi, Mg/+). The maximum effect of hSTC on phosphate excretion was observed 60-80 minutes postinjection. Lesser effects were obtained with higher and lower dosages of hormone. When renal cortical brush-border membrane vesicles were isolated from control and hormone-treated animals 80 minutes postinjection, the rate of Na+/Pi cotransport was found to be 40% higher in vesicles from hormone-treated animals (p < 0.01; 5 nmol hSTC/kg). Together, the renal clearance and membrane vesicle data indicate that hSTC participates in the renal regulation of Pi homeostasis in mammals.

Salmon calcitonin inhibits whole body Ca2+ uptake in young rainbow trout.

Gill Ca2+ transport (GCAT) in fish is regulated by a number of different hormones. Stanniocalcin (STC) from the corpuscles of Stannius (CS) is an inhibitor of GCAT, whereas pituitary-derived prolactin and cortisol stimulate GCAT. Other than this, however, little is known about the effects of other hormones on this important transport process. The role of calcitonin (CT) in calcium homeostasis in fish is still controversial. Whereas many studies have shown significant effects of CT on plasma calcium levels, an equal number of studies have failed to find any correlations between plasma calcium and CT levels in fish. Previous in vitro studies have shown that salmon CT has potent inhibitory effects on GCAT in isolated, perfused fish gill preparations, a finding that has never been corroborated in vivo. Therefore, in this report we examined the effects of salmon CT on whole body 45Ca uptake (as a measure of GCAT) in young rainbow trout. In support of the in vitro findings, we found that CT had significant inhibitory effects on GCAT. In parallel studies, we found that CT had no effects on STC secretion and only modest, stimulatory effects on STC mRNA levels in cultured trout CS cells. These finding suggest that both CT and STC function as negative regulators of GCAT in fish.

Bovine placental lactogen is a potent stimulator of growth and displays strong binding to hepatic receptor sites of coho salmon.

Juvenile coho salmon were treated with bovine placental lactogen (bPL) and bovine growth hormone (bGH) to examine the growth promoting activities of these proteins in a lower vertebrate. Fish were intraperitoneally injected either with 0.5 or 5.0 micrograms/g bPL or with 5.0 micrograms/g bGH once a week for 5 weeks. After only a single injection and 1 week of growth, the high dose of bPL stimulated a significant increase in weight and length relative to untreated fish or fish treated with a control protein, bovine serum albumin. At the end of the experiment, all hormone-treated groups were significantly larger than controls. Fish treated with 5 micrograms/g bPL gained more than three times as much weight as controls. The 5.0 micrograms/g bGH group grew at the same rate as fish treated with one-tenth this dose of bPL, indicating that bPL is a potent stimulator of growth in this species. Radioreceptor assays performed on coho salmon liver membrane preparations indicate that bPL binds with approximately 430-fold higher affinity than bGH, and some 8000-fold higher affinity than bovine prolactin. The action of bPL relative to the structure and function of salmonid pituitary hormones is discussed.

Calcium regulates stanniocalcin mRNA levels in primary cultured rainbow trout corpuscles of stannius.

Stanniocalcin (STC) is an inhibitor of gill calcium transport produced by the corpuscles of Stannius (CS), endocrine glands in bony fishes. In previous studies we have described how STC secretion is regulated by calcium both in vitro and in vivo, using rainbow trout as a model system. In this report we have examined the effects of calcium on STC mRNA levels in primary cultured trout CS cells. The results show that message levels are positively regulated by extracellular calcium concentrations within the physiological range. The calcium response was also temporally-related as more prolonged exposures tended to have greater effects. Similar concentrations of magnesium had no effect on message levels. This represents another level at which calcium regulates the CS cell, in addition to its established effects on STC synthesis and secretion. The results are discussed in relation to the other known calciotropic hormones, calcitonin and parathyroid hormone.

Evidence for two intracellular calcium pools in Dictyostelium: the cAMP-induced calcium influx is directed into a NBD-Cl- and 2,5-di-(tert-butyl)1,4-hydroquinone-sensitive pool.

Signal transduction in Dictyostelium for oriented movement and differentiation involves a fine tuning of the cytosolic Ca2+ concentration. We have previously shown that cAMP binding to the cell surface receptor elicits two cellular events: (i) to enhance Ca2+ entry across the plasma membrane; (ii) to increase Ca2+ uptake into Ca(2+)-sequestering organelles. Here we used permeabilised cells to show that cAMP-induced Ca2+ uptake in these cells was sensitive to the Ca2+ transport ATPase blocker 2,5-di-(tert-butyl)-1,4-hydroquinone (BHQ) and the vacuolar H(+)-ATPase inhibitor NBD-Cl. By contrast, bafilomycin A1 and vanadate, inhibitors of Ca2+ uptake into acidosomes in Dictyostelium, did not reduce the cAMP-induced Ca2+ uptake of permeabilised cells. GTP gamma S served as a tool to measure Ins(1,4,5)P3- (InsP3)-sensitive Ca2+ release. Following NBD-Cl or BHQ treatment Ca2+ release was reversibly inhibited. We conclude that the cAMP-controlled Ca2+ influx is directed into a NBD-Cl and BHQ-sensitive compartment, which comprises the InsP3-releasable pool. The acidosomal Ca2+ store seems to provide for additional Ca2+ if required.

Effect of oxygen on ascorbic acid uptake and concentration in embryonic chick brain.

The effects of oxygen on ascorbic acid concentration and transport were studied in chick embryo (Gallus gallus domesticus). During normoxic incubations, plasma ascorbic acid concentration peaked on fetal day 12 and then fell, before increasing again on day 20 when pulmonary respiration began. In contrast, cerebral ascorbic acid concentration rose after day 6, was maintained at a relatively high level during days 8-18, and then fell significantly by day 20. Exposure of day 16 embryos for 48 h to 42% ambient O2 concentration decreased ascorbic acid concentration by four-fifths in plasma and by one-half in brain, compared to values in normoxic (21% O2) or hypoxic (15% O2) controls. Hyperoxic preincubation of embryos also inhibited ascorbic acid transport, as evidenced by decreased initial rates of saturable and Na(+)-dependent [14C]ascorbic acid uptake into isolated brain cells. It may be concluded that changes in ascorbic acid concentration occur in response to oxidative stress, consistent with a role for the vitamin in the detoxification of oxygen radicals in fetal tissues. However, changing O2 levels have less effect on ascorbic acid concentration in brain than in plasma, indicating regulation of the vitamin by brain cells. Furthermore, the effect of hyperoxia on cerebral vitamin C may result, in part, from inhibition of cellular ascorbic acid transport.

[Elaboration and examination of the functioning of a new system for collection and analysis of information on foodborne and waterborne diseases in Poland]. / Opracowanie i sprawdzenie funkcjonowania nowego systemu gromadzenia i analizy danych na temat zatruc i zakazen pokarmowych w Polsce.

During 1986-1989 the new computerized system of collection and analysis of foodborne and waterborne infections and intoxications (based on the proposition of World Health Organization) was developed in the Department of Epidemiology of the National Institute of Hygiene in Warsaw. System consists of the new forms (including computer's form) and adapted original programming. The new forms were officially introduced by the Ministry of Health and Welfare for the use by the all Sanitary Stations in Poland from the beginning of 1991. That system was served to present data on epidemiological situation of foodborne and waterborne infections and intoxications in Poland in 1988-1991, demonstrated in the paper. Criteria of analysis were adapted to meet conditions of the international cooperation, and-first of all-to the requirements of Polish epidemiological situation.

[The effect of amphoteric tensides on the pharmaceutic availability of model drugs according to formulation]. / Badanie wplywu amfotenzydów na dostepnosc farmaceutyczna modelowych substancji leczniczych z postaci leku.

Pharmaceutical availability of chloramphenicol from o/w emulsion ointment and salicylic acid from tablets as influenced by the kind and concentration of amphoteric tenside has been investigated. It has been shown that amphoterics investigated variously influenced drug release, what makes possible to obtain the form of drug showing desired rate of release.

Ascorbate uptake by ROS 17/2.8 osteoblast-like cells: substrate specificity and sensitivity to transport inhibitors.

Ascorbate (reduced vitamin C) is required for bone formation. We have shown previously that both the osteoblast-like cell line ROS 17/2.8 and primary cultures of rat calvarial cells possess a saturable, Na(+)-dependent uptake system for L-ascorbate (J Membr Biol 111:83-91, 1989). The purpose of the present study was to investigate the specificity of this transport system for organic anions and its sensitivity to transport inhibitors. Initial rates of ascorbate uptake were measured by incubating ROS 17/2.8 cells with [L-14C]ascorbate at 37 degrees C. Uptake of [L-14C]ascorbate (5 microM) was inhibited 98 +/- 1% by coincubation with unlabeled L-ascorbate (3 mM) and 48 +/- 4% by salicylate (3 mM), but it was not affected by 3 mM formate, lactate, pyruvate, gluconate, oxalate, malonate, or succinate. Uptake of the radiolabeled vitamin also was not affected by acute (1 minute) exposure of the cells to the Na+ transport inhibitors amiloride and ouabain or the glucose transport inhibitor cytochalasin B. In contrast, anion transport inhibitors rapidly (less than 1 minute) and reversibly blocked [L-14C]ascorbate uptake. In order of potency, these drugs were 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) approximately equal to sulfinpyrazone greater than furosemide approximately equal to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). These findings indicate that the ascorbate transporter is relatively specific for the ascorbate anion, since other organic anions (with the exception of salicylate) did not compete with ascorbate for uptake. Rapid and reversible inhibition by the impermeant antagonists DIDS and SITS suggests that they interact directly with the ascorbate transporter, consistent with location of the transport system in the plasma membrane.

Mutant analysis suggests that cyclic GMP mediates the cyclic AMP-induced Ca2+ uptake in Dictyostelium.

Previous work has shown that streamer F (stmF) mutants of Dictyostelium discoideum exhibit prolonged chemotactic elongation in aggregation fields. The mutants carry an altered structural gene for cyclic GMP phosphodiesterase resulting in low activities of this enzyme. Chemotactic stimulation by cyclic AMP causes a rapid transient increase in the cyclic GMP concentration followed by association of myosin heavy chains with the cytoskeleton. Both events persist several times longer in stmF mutants than in the parental strain, indicating that the change in association of myosin with the cytoskeleton is transmitted directly or indirectly by cyclic GMP. We measured the cyclic AMP-induced Ca2+ uptake with a Ca(2+)-sensitive electrode and found that Ca2+ uptake was prolonged in stmF mutants but not in the parental strain. The G alpha 2 mutant strain HC33 (fgdA), devoid of InsP3 release and receptor/guanylate cyclase coupling, lacked Ca2+ uptake. However, the latter response and cyclic GMP formation were normal in the signal-relay mutant strain agip 53 where cyclic AMP-stimulated cyclic AMP synthesis is absent. LiCl, which inhibits InsP3 formation in Dictyostelium, blocked Ca2+ uptake in a dose-dependent manner. The data indicate that the receptor-mediated Ca2+ uptake depends on the InsP3 pathway and is regulated by cyclic GMP. The rate of Ca2+ uptake was correlated in time with the association of myosin with the cytoskeleton, suggesting that Ca2+ uptake is involved in the motility response of the cells.

Evidence for electrogenic sodium-dependent ascorbate transport in rat astroglia.

The dependence of ascorbate uptake on external cations was studied in primary cultures of rat cerebral astrocytes. Initial rates of ascorbate uptake were diminished by lowering the external concentrations of either Ca2+ or Na+. The Na(+)-dependence of astroglial ascorbate uptake gave Hill coefficients of approximately 2, consistent with a Na(+)-ascorbate cotransport system having stoichiometry of 2 Na+:1 ascorbate anion. Raising external K+ concentration incrementally from 5.4 to 100 mM, so as to depolarize the plasma membrane, decreased the initial rate of ascorbate uptake, with the degree of inhibition depending on the level of K+. The depolarizing ionophores gramicidin and nystatin slowed ascorbate uptake by astrocytes incubated in 5.4 mM K+; whereas, the nondepolarizing ionophore valinomycin did not. Qualitatively similar results were obtained whether or not astrocytes were pretreated with dibutyryl cyclic AMP (0.25 mM for 2 weeks) to induce stellation. These data are consistent with the existence of an electrogenic Na(+)-ascorbate cotransport system through which the rate of ascorbate uptake is modulated by endogenous agents, such as K+, that alter astroglial membrane potential.

Substrate regulation of ascorbate transport activity in astrocytes.

Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-[14C]ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-[14C]ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-[3H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.