Optics research at the U.S. Army Research Laboratory.

The U.S. Army Research Laboratory (ARL) is the Army's premier laboratory for land forces. The Army relies on ARL for scientific discoveries, technological advances, and analyses that enable capabilities a future Army will need to persevere over adversaries. Although a relatively young organization that will celebrate 25 years of the discovery, innovation, and transition of science and technology in October 2017, ARL has already had significant impact in a wide range of scientific and technological disciplines. In this paper, we highlight some of its past and recent achievements in optics and photonics.

Adenoviral transfer of human aquaporin-1 gene to rat liver improves bile flow in estrogen-induced cholestasis.

Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per µmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data suggest that the adenoviral transfer of hAQP1 gene to the livers of EE-induced cholestatic rats improves bile flow by enhancing the AQP-mediated bile salt-induced canalicular water secretion. This novel finding might have potential therapeutic implications for cholestatic diseases.

Extended cold storage of cultured hepatocytes impairs endocytic uptake during normothermic rewarming.

During hypothermic preservation of cells (0-4°C), metabolism is diminished and energy-dependent transport processes are arrested. The effect of hypothermic preservation of hepatocytes in endocytic transport following rewarming has not been previously reported. We evaluated the uptake of EGF (Epidermal Growth Factor) ligand conjugated to fluorescent Quantum Dots (QDs) probes in rat hepatocytes after 24 and 72h cold storage in University of Wisconsin (UW) solution at 4°C. QDs uptake was visualized during rewarming to 37°C under air or, in a second approach, at the end of rewarming under 5% CO2. After 24h in UW solution, QDs were internalized under both rewarming conditions similar to non-preserved hepatocytes and cells maintained a normal cytoskeleton distribution. However, in hepatocytes preserved 72h none of the cells internalized QDs, which remained bound to the membranes. After rewarming, this group showed diminished actin staining and 60% reduction in ATP levels, while viability was maintained at ∼70%. Our results present evidence that, hypothermic preservation for 72h in UW solution at 4°C does not prevent EGFR (epidermal growth factor receptor) activation but irreversibly impairs endocytic uptake upon EGF stimulation; presumably due to actin cytoskeleton disassembling besides reduced ATP pool. Our approach can be applied on other membrane receptor systems and with other hypothermic preservation solutions to understand the effect of cooling in endocytic transport and to determine the optimal cold storage period.

Impaired localisation and transport function of canalicular Bsep in taurolithocholate induced cholestasis in the rat.

BACKGROUND: Taurolithocholate induced cholestasis is a well established model of drug induced cholestasis with potential clinical relevance. This compound impairs bile salt secretion by an as yet unclear mechanism. AIMS: To evaluate which step/s of the hepatocellular bile salt transport are impaired by taurolithocholate, focusing on changes in localisation of the canalicular bile salt transporter, Bsep, as a potential pathomechanism. METHODS: The steps in bile salt hepatic transport were evaluated in rats in vivo by performing pharmacokinetic analysis of (14)C taurocholate plasma disappearance. Bsep transport activity was determined by assessing secretion of (14)C taurocholate and cholyl-lysylfluorescein in vivo and in isolated rat hepatocyte couplets (IRHC), respectively. Localisation of Bsep and F-actin were assessed both in vivo and in IRHC by specific fluorescent staining. RESULTS: In vivo pharmacokinetic studies revealed that taurolithocholate (3 micro mol/100 g body weight) diminished by 58% canalicular excretion and increased by 96% plasma reflux of (14)C taurocholate. Analysis of confocal images showed that taurolithocholate induced internalisation of Bsep into a cytosolic vesicular compartment, without affecting F-actin cytoskeletal organisation. These effects were reproduced in IRHC exposed to taurolithocholate (2.5 micro M). Preadministration of dibutyryl-cAMP, which counteracts taurolithocholate induced impairment in bile salt secretory function in IRHC, restored Bsep localisation in this model. Furthermore, when preadministered in vivo, dibutyryl-cAMP accelerated recovery of both bile flow and bile salt output, and improved by 106% the cumulative output of (14)C taurocholate. CONCLUSIONS: Taurolithocholate impairs bile salt secretion at the canalicular level. Bsep internalisation may be a causal factor which can be prevented by dibutyryl-cAMP.

Beneficial effects of silymarin on estrogen-induced cholestasis in the rat: a study in vivo and in isolated hepatocyte couplets.

The effect of silymarin (SIL) on 17alpha-ethynylestradiol (EE)-induced cholestasis was evaluated in rats. EE (5 mg/kg, subcutaneously, daily, for 5 days) decreased both the bile-salt-dependent and the bile-salt-independent fractions of the bile flow. The decrease in the former was associated to a reduction in the bile salt pool size (-58%), and this effect was completely prevented by SIL. This compound also counteracted the inhibitory effect induced by EE on HCO(3)(-) but not glutathione output, 2 major determinants of the bile-salt-independent bile flow. EE decreased the secretory rate maximum (SRM) of tauroursodeoxycholate, (-71%) and bromosulfophthalein (BSP; -60%), as well as the expression of the BSP canalicular carrier, mrp2; SIL failed to increase mrp2 expression, and had only a marginal beneficial effect on both tauroursodeoxycholate and BSP SRM values. However, the two-compartment model-based kinetic constant for BSP canalicular transfer was significantly improved by SIL (+262%). SIL decreased rather than increased CYP3A4, the cytochrome P450 isoenzyme involved in the oxidative metabolism of EE, and had no inhibitory effect on the UDP-glucuronosyltrasferase isoforms involved in the formation of its 17beta-glucuronidated, more cholestatic metabolite. Pretreatment of isolated rat hepatocyte couplets with silibinin, the major, active component of SIL, counteracted the estradiol 17beta-glucuronide-induced decrease in the percentage of couplets secreting apically the fluorescent bile acid analogue, cholyl-lysyl-fluorescein. These results show that SIL protects against EE-induced cholestasis by normalizing mainly the decrease in the bile salt pool size and HCO(3)(-) output, and probably by counteracting the cholestatic effect of its cholestatic, glucuronidated metabolite.

Molecular basis of perinatal changes in UDP-glucuronosyltransferase activity in maternal rat liver.

The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform belonging to family 2, was analyzed in rat liver. Nonpregnant, pregnant (19-20 days of pregnancy), and two groups of postpartum animals corresponding to early and middle stages of lactation (2-4 and 10-12 days after delivery, respectively) were studied. UGT activity determined in UDP-N-acetylglucosamine-activated microsomes revealed that bilirubin, p-nitrophenol, and ethynylestradiol (17beta-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery. p-Nitrophenol, androsterone, and estrone conjugation rate increased in postpartum rats. Western blot analysis performed with anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed decreased levels of all family 1 isoforms and UGT2B1 during pregnancy. In postpartum animals, protein level recovered (1A5 and 2B1) or even increased (1A1 and 1A6) with respect to control rats. Northern blot analysis suggested that expression of UGT proteins is down-regulated at a post-translational level during pregnancy and that increased levels of 1A1 and 1A6 observed in postpartum rats were associated to increased mRNA. To establish whether prolactin is involved in up-regulation of UGT1A1 and 1A6 postpartum, ovariectomized rats were treated with 300 microg of ovine prolactin per day for 7 days. The data indicated that prolactin was able to increase expression of UGT1A6 (protein and mRNA) but not 1A1. Thus, prolactin is the likely mediator of the increased expression of UGT1A6 observed in maternal liver postpartum.

[In vivo apoptotic effect of alpha-2b interferon (IFN) on rat preneoplastic liver ]. / Efecto apoptotico in vivo del interferon alfa-2b (IFN) sobre higados preneoplasicos de rata.

In order to know whether IFN alpha prevents in vivo oncogenesis in the very-early-stage cancer cells, we evaluated the action of IFN alpha-2b on preneoplastic foci in rats. Animals were divided into six groups: subjected to an initiation-promotion model of cancer development (G1), treated with IFN alpha-2b during: a) initiation-promotion (G2), b) initiation (G3), promotion (G4); subjected only to an initiation stage (G5) and treated with IFN alpha-2b during this period (G6). The number and area of rGST P-positive foci were reduced and the Apoptotic index was increased in G2, 3 and 6. Bcl-2 and Bcl-XL protein levels were decreased in IFN alpha-2b-treated rats. Increased levels of mitochondrial Bax protein were observed in G2, 3 and 6. In conclusion, preneoplastic hepatocytes in the IFN alpha-2b-treated rats undergo programmed cell death as a result of a significant increase of Bax and its translocation to the mitochondria.

Efecto apoptotico in vivo del interferon alfa-2b (IFN) sobre higados preneoplasicos de rata. / [In vivo apoptotic effect of alpha-2b interferon (IFN) on rat preneoplastic liver ]

In order to know whether IFN alpha prevents in vivo oncogenesis in the very-early-stage cancer cells, we evaluated the action of IFN alpha-2b on preneoplastic foci in rats. Animals were divided into six groups: subjected to an initiation-promotion model of cancer development (G1), treated with IFN alpha-2b during: a) initiation-promotion (G2), b) initiation (G3), promotion (G4); subjected only to an initiation stage (G5) and treated with IFN alpha-2b during this period (G6). The number and area of rGST P-positive foci were reduced and the Apoptotic index was increased in G2, 3 and 6. Bcl-2 and Bcl-XL protein levels were decreased in IFN alpha-2b-treated rats. Increased levels of mitochondrial Bax protein were observed in G2, 3 and 6. In conclusion, preneoplastic hepatocytes in the IFN alpha-2b-treated rats undergo programmed cell death as a result of a significant increase of Bax and its translocation to the mitochondria.

Effect of silymarin on biliary bile salt secretion in the rat.

The effect of the hepatoprotector silymarin on bile secretion, with particular regard to bile salt secretion, was studied in Wistar rats. Silymarin (25, 50, 100, and 150 mg/kg/day, i.p., for 5 days) induced a dose-dependent increase in bile flow and bile salt secretion, the maximal effect being reached at a dose of 100 mg/kg/day (+17 and +49%, for bile flow and bile salt output, respectively; P < 0.05). Assessment of bile salt composition in bile revealed that stimulation of the bile salt secretion was accounted for mainly by an increase in the biliary secretion of beta-muricholate and, to a lesser extent, of alpha-muricholate, chenodeoxycholate, ursodeoxycholate, and deoxycholate. The maximum secretory rate (T(m)) of bile salts, as assessed by infusing the non-hepatotoxic bile salt tauroursodeoxycholate i.v. at stepwise-increasing rates, was not influenced by silymarin. The flavonolignan also increased the endogenous bile salt pool size (+53%, P < 0.05) and biliary bile acid excretion after bile acid pool depletion (+54%, P < 0.05), a measure of de novo bile salt synthesis. These results suggest that silymarin increases the biliary excretion and the endogenous pool of bile salts by stimulating the synthesis, among others, of hepatoprotective bile salts, such as beta-muricholate and ursodeoxycholate.

Induction of phase II biotransformation reactions in rat jejunum during lactation. Possible involvement of prolactin.

The effect of lactation on UDP-glucuronosyltransferase (UGT) and Glutathione S-transferase (GST) activities was studied in jejunum from mother rats, 14 (LM14) and 21 (LM21) days after delivery. p-Nitrophenol glucuronidation rate was increased in LM14 and LM21 rats while conjugation of bilirubin and estrone was not affected and androsterone glucuronidation was decreased. Additional studies, including Western blotting and microsomal lipid analysis, revealed that the enhancement in p-nitrophenol UGT activity is most likely associated with an inductive process rather than with a modification in enzyme constraint. GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was also increased in LM14 and LM21 while activity towards 1,2-dichloro-4-nitrobenzene (DCNB) was not affected. Western blotting revealed a significant increase in the cytosolic content of mu (rGSTM2) and pi (rGSTP1) class subunits in LM14 and LM21 groups, while the alpha class subunit rGSTA2 remained unchanged. To evaluate the potential modulatory role of prolactin on the same enzyme systems, ovariectomized rats were treated with ovine prolactin (oPRL) at doses of 100, 200 and 300 microg/100 g body wt. per day for 4 days. Hormone administration affected UGT activities towards p-nitrophenol and androsterone and GST activity towards CDNB in a way and magnitude consistent with those produced in lactating rats, while conjugation of estrone, bilirubin and DCNB were unchanged. Western blotting data were also consistent with those of lactating rats. These results indicate that UGT and GST activities are increased in rat jejunum during lactation, due to induction of some specific isoforms, and that prolactin is the likely mediator of these effects.

Taurocholate-induced inhibition of hepatic lysosomal degradation of horseradish peroxidase.

Endocytosed proteins in hepatocytes are transported to lysosomes for degradation. Metabolites accumulating in these organelles are released into bile by exocytosis, a process that seems to be regulated by the bile salt taurocholate (TC). In this study we examined if TC is also involved in the control of the lysosomal degradation of endocytosed proteins. We used [(14)C]sucrose-labeled horseradish peroxidase ([(14)C]S-HRP), a probe suitable to evaluate lysosomal proteolysis. TC-infused rats as well as isolated rat hepatocytes exposed to TC showed a significant inhibition in the lysosomal degradation of [(14)C]S-HRP (approximately 30%), with no change in either the uptake or the amount of protein reaching lysosomes. Under these conditions, the in vitro assay of lysosomal cathepsins B, L, H, and D revealed no change in their activities, suggesting that a reversible inhibition (lysosomal alkalinization?) was taking place in hepatocytes. Nevertheless, lysosomal pH measured using fluorescein isothiocyanate-dextran was shown not to be altered by TC. In addition, TC was unable to inhibit proteolysis in [(14)C]S-HRP loaded lysosomes or interfere in cathepsin assays. The results suggest that TC inhibits the lysosomal degradation of endocytosed proteins in hepatocytes and that the mechanism does not involve an effect of the bile salt per se or a rise in lysosomal pH.

Role of bile salts in colchicine-induced hepatotoxicity. Implications for hepatocellular integrity and function.

Colchicine, a microtubule-disrupting agent, induces hepatotoxicity in experimental animals at the doses commonly employed to explore vesicular transport in the liver. The effect of manipulations of the bile salt pool on colchicine-induced hepatotoxicity was studied in rats to determine the role of bile salts in this phenomenon. Leakage of enzyme markers of liver-cell damage into plasma and bile induced by colchicine pre-treatment displayed a sigmoidal log dose-effect curve, the half-maximal effect being reached at 0.12 micromol per 100 g body wt. Lumicolchicine, instead, showed no harmful effect. Maximal increment of biliary LDH discharge induced by colchicine was reduced from 950 +/- 124% to 216 +/- 29% by bile diversion leading to a marked reduction in bile salt output, and this parameter was further decreased to 100 +/- 13% and 157 +/- 39% by subsequent repletion of the bile salt pool with the hydrophilic bile salts taurodehydrocholate and tauroursodeoxycholate, respectively. Conversely, infusion of taurocholate into non-bile salt depleted, colchicine-treated rats led to cholestasis and massive discharge of enzymes into both blood and bile. Our data show conclusively that colchicine-induced hepatotoxicity depends on the magnitude and composition of the bile salt flux traversing the liver. They also support the view that functional integrity of vesicular mechanisms presumably involved in membrane repair are indispensable to protect the hepatocytes from the damaging effect of bile salts during normal bile formation.

Taurolithocholate can inhibit the biliary discharge of lysosomes in the rat.

The natural bile salt taurolithocholate (TLC) impairs the biliary excretion of lipids and proteins, which are known to reach the canaliculus via vesicles. In this study we examined whether these observations could be extended to the exocytic discharge of lysosomal contents into bile. The single intravenous injection of a cholestatic dose of TLC, 3 micromol/100 g body wt., markedly inhibited the biliary excretion of the lysosomal enzymes acid phosphatase and beta-glucuronidase, despite the excretion of bile salts being normalized after a transient diminution. Under such a condition, TLC did not affect the normal transport to and the processing in lysosomes of the exogenously administered [14C]sucrose-labeled horseradish peroxidase. However, the biliary excretion of the radioactive lysosomal metabolites of the protein was significantly reduced. The results indicate that TLC can inhibit the biliary discharge of lysosomes in the rat without altering the functional integrity of these organelles. Possible explanations for these findings are discussed.

Assessment of the in vivo hepatic lysosomal processing of horseradish peroxidase.

The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its lysosomal proteolysis and the biliary output of its possible lysosomal metabolites by rat liver in vivo. HRP was covalently linked to [14C]sucrose to provide a label that remains trapped within lysosomes after proteolysis. The [14C]sucrose-labelled HRP was injected into the portal vein of rat, and after 30 min about 34% of the injected radiolabel was present in the liver. Subcellular fractionation by differential centrifugation and further purification of lysosomes in a Percoll gradient showed that radiolabel was concentrated in lysosomes and indicated that about 91% of the total proteolysis of HRP in liver could be attributed to these organelles. The in vivo lysosomal degradation rate of HRP at 30 min was about 40%/h, decreasing over time. The lysosomal inhibitors chloroquine and leupeptin suppressed proteolysis of HRP by about 30 and 60%, respectively. Analysis of the 14C excreted in bile by trichloroacetic acid precipitation and by SDS-polyacrylamide gel electrophoresis showed a minor fraction, which was intact HRP (40 kDa), and a major fraction, which was associated with material smaller than 3 kDa. The biliary output of these low molecular mass products, in contrast to that of intact HRP, did not gradually decline with time and represented about 3% of the corresponding amounts in liver. Chloroquine and leupeptin specifically decreased their biliary excretion (about 60%), giving additional support to their lysosomal origin. In addition, the overall hepatic processing of [14C]sucrose-labelled HRP did not differ from that of the native HRP measured by enzyme assay, indicating no significant alteration caused by the labelling procedure.

Taurolithocholate-induced inhibition of biliary lipid and protein excretion in the rat.

Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 mumol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.

Case report: acute appendicitis in an inguinal hernia.

Primary appendicitis presenting in a hernia sac is uncommon. Diagnosis depends on a high index of suspicion. The authors present a case report of a 65-year-old male with a two-day history of a painful irreducible right inguinal mass; he denied abdominal pain, nausea, vomiting, fever, or chills.

Hepatic handling of photoirradiated bilirubin. A study in isolated perfused Wistar rat liver.

Conjugation has been considered the rate-limiting step for bilirubin hepatic transport, and bypass of this metabolic step could explain why photobilirubins can be rapidly cleared by the liver. In this paper we assessed whether photoirradiation may enhance the bilirubin overall hepatic transport in the isolated perfused Wistar rat liver, a model possessing intact transport and conjugating systems. Bilirubin was administered as a bolus so as to reach a perfusate concentration of approximately 10 microM (bilirubin/albumin molar ratio 1:17). Perfusate light exposure (0.56.10(15) quanta s-1 cm-2) yielded 7-10% of configurational photoisomers, which were further identified as (4Z,15E/4E,15Z)-bilirubin IX alpha. Under such conditions, the perfusate removal rate was increased by 39% over that from dark conditions. Likewise, biliary excretion, estimated as total bilirubin recovery at 60 min, was also increased (+48%). This later improvement was mainly produced at the expense of unconjugated bilirubin, which most likely derived from its configurational photoisomers that, once excreted into bile, readily re-isomerized to the parent compound. In addition, this increment was partially due to a delayed improvement of monoglucuronide pigment excretion. The calculated hepatic pigment content was significantly higher under light conditions. A direct assessment of hepatic content of different bilirubin moieties at 20 min after bilirubin administration confirmed that such an increment was fully accounted for by unconjugated pigment. Our finding that hepatic pigment content rose (despite a higher biliary excretion) when the bilirubin was irradiated suggests a higher net uptake of photoisomers than native pigment. This observation, and the finding that bilirubin photoisomers were usually excreted without undergoing conjugation even if the metabolic system is active, contribute to explain the greater appearance of unconjugated bilirubin in Wistar rat bile under light exposure.

Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes.

1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.

Impairment of bile secretion induced by aflatoxin B1 in the rat.

The influence of Aflatoxin B1 on bile secretion was examined in rats. The toxin was given i.p. at a dose of 100 micrograms/kg body wt. It was observed 48 h after the injection a decrease in bile acid-independent and bile acid-dependent flow. Bile acid secretion was also decreased, whereas the outputs of cholesterol and phospholipids and the cholesterol saturation index of bile were unchanged. (14C) erythritol and (14C) sucrose biliary clearances decreased in parallel to bile flow, suggesting that the decrease in bile flow was of canalicular origin and that there was not alteration in the permeability of the biliary system.

Effect of spironolactone on bilirubin metabolism in rat liver and small intestinal mucosa.

In vitro and in vivo experimental models were designed for the study of the effect of spironolactone (SP) on bilirubin metabolism in rat liver and small intestinal mucosa. In vitro studies included uptake of bilirubin by liver slices and intestinal sheets, determination of glucuronyltransferase activity in mucosal homogenates, and the handling of bilirubin by the isolated perfused liver after bilirubin overload. In vitro studies were carried out to measure the plasma disappearance rate of bilirubin and to determine the extent of bilirubin conjugation and biliary excretion of the pigment infused intravenously. The results obtained suggested that the mechanisms involved in the uptake of bilirubin by tissues were not influenced by SP pretreatment. Glucuronyltransferase activity in the small intestinal mucosa was significantly induced by SP, as previously observed in rat liver. Isolated perfused livers from SP-treated rats, as well as treated living rats, exhibited a greater than normal capacity for bilirubin excretion into bile at the expense of bilirubin diglucuronide. Conjugated bilirubin in the small intestinal mucosa of rats infused with unconjugated pigment was also increased after SP pretreatment. The results favoured the conclusion that SP is an inducer of bilirubin conjugation in the livers as well as in extrahepatic tissues, such as the small intestinal mucosa.