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The aging olfactory epithelium: neurogenesis, response to damage, and odorant-induced activity

Loo,A., S.Youngentob, P.Kent & J.Schwob
Int. J. Dev. Neurosci. (1996) 14(7/8):881-900 [Back to Resume]

Olfactory epithelium retains the capacity to recover anatomically after damage well into adult life and perhaps throughout its duration. None the less, olfactory dysfunctions have been reported widely for elderly humans. The present study investigates the effects of aging on the neurophysiological and anatomical status of the olfactory epithelium in barrier-raised Fischer 344X Brown Norway F1 hybrid rats at 7, 10, 25 and 32/35 months old. The posterior part of the olfactory epithelium in 32/35-month-old rats is well preserved. Globose basal cells are dividing, and new neurons are being born even at this advanced age. None the less, the numbers of proliferating basal cells and immature, GAP-43 (+) neurons are significantly decreased. Neurophysiological status was evaluated using voltage-sensitive dye techniques to assess inherent patterns of odorant-induced activity in the epithelium lining the septum and the medial surface of the turbinates. In middle and posterior zones of the epithelium, there were neither age-related changes in overall responsivity of this part of the olfactory epithelium to any of five odorants, nor shifts in the location of the odorant-induced hotspots. The inherent activity patterns elicited by the different odorants do become more distinct as a function of age, which probably reflects the decline in immature neurons and a slight, but not statistically significant, increase in mature neurons as a function of age. In contrast with the excellent preservation of posterior epithelium, the epithelium lining the anterodorsal septum and the corresponding face of the turbinates is damaged in the 32/35-month-old animals: in this part, horizontal basal cells are reactive, more basal cells and sustentacular cells are proliferating than in younger animals or in posterior epithelium of the same animals, and the neuronal population is less mature on average. Our findings indicate that degeneration of the olfactory epithelium is not an inevitable or pre-programmed consequence of the aging process, since the posterior zone of the epithelium is very well preserved in these barrier-protected animals. However, the deterioration in the anterior epithelium suggests that environmental insults can accumulate or become more severe with age and overwhelm the regenerative capacity of the epithelium. Alternatively, the regenerative capacity of the epithelium may wane somewhat with age. Either of these mechanisms or some combination of them can account for the functional and anatomical deterioration of the sense of smell associated with senescence in humans.

Parallelism of serum HCV-RNA and ALT levels during breakthrough and/or relapse in patients treated with alpha-interferon for chronic hepatitis C

Reedy, D.W., J.Nandi, A.Loo, H.Mahoney & R.A.Levine
Hepatology (1996) 24(4)Pt 2:565A [Back to Resume]

Some patients with chronic hepatitis C (CHC) infection normalize their ALT during and/or after interferon therapy only to experience a subsequent rise in ALT. The nature of these phenomena, considered a breakthrough (BT) or, after cessation of therapy, a relapse (RL), are unknown. Possible factors include emergence of variant strains of HCV, development of neutralizing antibodies to alpha-interferon or compromised immune response. We studied 29 non-cirrhotic patients who were treated with a de-escalating dose of Roferon or Intron-A, 6 MIU tiw for 3 months then 3 MIU tiw for the next 3-9 months. Patients whose ALT did not normalize within 3 months of initiating therapy were considered nonresponders and dropped from the study. Serum was drawn monthly for ALT and quantitative HCV-RNA by PCR (AMPLICOR-Roche). Results: At the start of therapy HCV-RNA was detectable (>500 copies/ml) in all patients with a mean of 420,518. 7 (24%) and 5 (17%) patients experienced BT and RL, respectively. HCV-RNA levels were undetectable prior to 4/7 BT and 5/5 RL, respectively. A rise in HCV-RNA preceded an ALT rise by >1 month in 25% BT and 40% RL. ALT elevations were associated with simultaneous rise in HCV-RNA in 3/7 BT and 4/5 RL. HCV-RNA remained undetectable in 2/7 BT and 0/5 RL. After BT or RL, undetectable viral load and normal ALT were re-established in 35% of patients, 3/7 BT and 5/5 RL patients, respectively, were complete responders (CR), defined as normal ALT level at the end of treatment, and 4 of these 12 patients developed a sustained CR. 9/29 patients (31%) without a BT or RL had CR to interferon. Conclusions: BT and RL showed parallelism of serum HCV-RNA and ALT levels, suggesting that ALT alone may reflect viral load. Concordance of these 2 parameters is common while disparity is rare during and/or after alpha-interferon therapy of CHC. Supported in part by Schering Corporation and Hoffman LaRoche, Inc.

Dietary supplementation of nucleotides and arginine promotes healing of small bowel ulcers in experimental enterocolitis

Sukumar,P., A.Loo, E.Magur, J.Nandi, A.Oler & R.A.Levine
Dig. Dis. Sci. (1997) 42:1530-1536 [Back to Resume]

We previously showed that intravenous total parenteral nutrition supplemented with nucleosides and nucleotides (NS/NT) promoted ulcer healing in rats with indomethacin-induced ileitis. The present study evaluated whether dietary NT supplementation would similarly affect ulcer healing in this model. Female Lewis rats were randomized into either control or experimental groups receiving yeast RNA containing NT or arginine, glutamine, fish oil, guar gum, or a combination of yeast RNA+arginine diets. Ileitis was induced by two doses of indomethacin (7.5 mg/kg) administered subcutaneously 24 hr apart. Ulcer number and length were determined at 4, 8, and 14 days after induction of ileitis. Ileal villous and crypt length, crypt-villous ratio, and bromodeoxyuridine (BrdU) labeling were studied in the control and yeast RNA-supplemented diet groups. Ileal ulceration was present in all groups at 4 and 8 days and was almost healed by 14 days. Rats receiving yeast RNA, arginine, and yeast RNA + arginine diets showed a significant decrease in ulcer number (56%, 28%, and 34%, respectively) and length (67%, 41%, and 48%, respectively) compared to controls at 8 but not at 4 days. Glutamine, fish oil, and guar gum had no effect on ulcer healing at 4, 8, or 14 days. Among the histological parameters, a significant decrease in crypt length in the yeast RNA-supplemented group at 8 days suggested an acceleration of the healing process and restoration to a near-normal crypt-villous architecture. We conclude that the yeast RNA, arginine, and yeast RNA + arginine diets accelerated ulcer healing, as indicated by decreased ulcer number and length. We postulate that the underlying mechanism(s) contributing to ulcer healing may be related, in part, to increased cell proliferation.

An endogenous activator protein of H+,K+-ATPase regulates acid secretion in isolated rabbit gastric glands

Nandi,J., A.Loo & R.A.Levine
Gastroenterology (1997) 112(4):A231 [Back to Resume]

Our earlier studies demonstrated that an endogenous activator protein (AP) stimulates gastric H+,K+-ATPase activity in vitro. We postulated that AP may be a target for signal transduction of gastric H+ transport (J.Biol.Chem. 262:5664-70, 1987). The present study evaluated the direct role of AP on intracellular modulation of gastric H+ transport using an AP-specific antibody (AP-Ab). Methods: Monospecific polyclonal antibody was raised against a homogeneous preparation of AP in young female rabbits. Cytosolic and membrane fractions of rabbit parietal cells (PC) were electrophoresed on SDS-PAGE and analyzed by Western blot using polyclonal AP-Ab. The AP-Ab was subsequently tested for its effects on H+,K+-ATPase activity and H+ secretion in PC membrane fractions and permeabilized gastric glands (PGG), respectively. H+ secretion was measured by an indirect method using [14C] aminopyrine uptake ratio. Results: AP-Ab recognized primarily 2 closely apposed bands near the 40kDa region of the cytosolic fraction, but only one of these bands in the membraine fraction. In contrast, the 40kDa band was undetectable in a Triton-soluble membrane fraction, suggesting AP may be associated partly with the cellular cytoskeleton. The catalytic a and b subunits of H+,K+-ATPase in the membrane fraction at the 94kDa region was not recognized by AP-Ab. AP-stimulated H+,K+-ATPase activity was inhibited by increaing concentrations of AP-Ab without affecting basal (Mg2+-dependent) ATPase activity. Similarly, AP-Ab decreased histamine-stimulated aminopyrine uptake in a dose-dependent manner in PGG. Conclusions: The AP-Ab was found to be specific for AP, but not for the a and b subunits of H+,K+-ATPase. Since AP-Ab inhibited H+,K+-ATPase and histamine-stimulated aminopyrine uptake, this suggests, for the first time, direct evidence for a regulatory role of AP on gastric H+ transport.

AST/ALT ratio >=1 is not diagnostic of cirrhosis in patients with chronic hepatitis C

Reedy,D.W., A.Loo & R.A.Levine
Dig. Dis. Sci. (1998) 43(9):2156-2159 [Back to Resume]

Medical guidelines for interferon-alpha2a or -alpha2b (IFN-alpha) treatment of chronic hepatitis C virus (HCV) infection depend upon baseline liver histology. A better long-term response to IFN-alpha therapy correlates with less inflammation and absence of cirrhosis. It has been suggested that the presence of cirrhosis in patients with chronic hepatitis C virus infection may be predicted based on an AST/ALT ratio > or = 1. This study was designed to determine if the presence of cirrhosis can be predicted in patients with chronic HCV infection by such a ratio. Seventy-seven patients, including 23 cirrhotics, with chronic HCV infection were studied. Serum ALT, AST, and HCV-RNA levels and hepatic activity index (HAI), reflecting histologic inflammation in all liver biopsies, were assessed. AST/ALT ratios and mean ALT, AST, and HCV-RNA were determined for both cirrhotic and noncirrhotic patients. HAI was correlated with ALT, AST, and HCV-RNA levels, the latter determined by quantitative RT-PCR. The likelihood ratio (LR) and positive predictive value of an AST/ALT ratio > or = 1 for cirrhosis was 7.3 and only 77%, respectively. In cirrhotics vs noncirrhotics, there were no significant differences between mean serum ALT (149 +/- 28 vs 176 +/- 17 units/liter), AST (139 +/- 28 vs 102 +/- 8 units/liter), or HCV-RNA levels (589,160 +/- 147,053 vs 543,915 +/- 75,497 copies/ml), respectively. There was a significant, but clinically weak, correlation between serum ALT and HAI (r = 0.234), and none between HAI and either serum AST or HCV-RNA levels. Our results support the need for a liver biopsy prior to treatment of chronic HCV infection, since the AST/ALT ratio fails to predict accurately the presence of cirrhosis.

Expression and characterization of protein kinase C in isolated rabbit parietal cells

Nandi,J., A.Loo, S.Kim & R.A.Levine
Int. J. Molecular Medicine (1999) 3(5):521-526 [Back to Resume]

We investigated the expression, characterization and distribution of protein kinase C (PKC) isozymes in isolated rabbit parietal cells (PC). Cellular extracts of PC were analyzed by Western blot using isozyme-specific antibodies. The Ca2+-independent PKC-epsilon was detected in cytosolic, membrane and cytoskeletal fractions of basal and histamine-stimulated PC, whereas the Ca2+-dependent PKC-alpha was confined to the cytosolic and membrane fractions. Cytosolic and membrane fractions were partially purified by DEAE cellulose column chromatography with elution of increasing NaCl concentration. Eluates of 0.15 M and 0.3 M NaCl PC fractions were identified as PKC-alpha and -epsilon isoforms, respectively. Phorbol 12-myristate 13-acetate (TPA) treatment of PC for 15, 30 and 60 sec decreased significantly cytosolic PKC-alpha and increased membrane-associated PKC-alpha. In contrast to the distribution of PKC-alpha, TPA did not alter membrane or cytosolic level of PKC-epsilon. Comparison of the dose-response curves between TPA-induced hydrogen (H+) secretion, as measured by aminopyrine (AP) uptake, and the membrane-associated PKC-alpha suggests that translocation of PKC-alpha is not involved in the H+ secretory process in PC. Furthermore, a PKC inhibitor, staurosporine, produced a concentration-dependent enhancement of histamine-stimulated H+ secretion. These findings suggest that PKC-alpha plays a negative modulatory role, rather than an obligatory role, in H+ secretion. The localization and distribution of PKC-epsilon into the cytoskeletal fraction of PC also suggests that this isozyme may be involved in the cellular regulation of reversible morphological transformation during stimulation.

Dietary nucleotides augment dextran sulfate sodium- induced distal colitis in rats

Sukumar,P., A.Loo, R.Aldophe, J.Nandi, A.Oler & R.A.Levine
J. Nutrition (1999) 129:1377-1381 [Back to Resume]

We have previously shown that enteral and parenteral supplementation of nucleotides (NT) accelerates healing of small-bowel ulcers in rats with indomethacin-induced ileitis. The purpose of this study was to evaluate whether dietary NT supplementation would similarly affect ulcer healing in dextran sulfate sodium (DSS)-induced colitis in rats. Male Sprague-Dawley rats were randomly assigned to receive either nucleotide-free (NF) or NT-supplemented diets. After 2 d of prefeeding, colitis was induced by including 40 g/L of DSS in drinking water for 3 d, followed thereafter by tap water. Rats from each group were killed at 7 and 12 d after induction of colitis. Additional rats were also used for both the groups as controls (untreated groups). The length of colon was measured and evaluated by histological score. Colonic myeloperoxidase (MPO) activity was assessed. In a separate series of experiments, rats were studied at 0, 4, 7, and 12 d for interleukin-1beta (IL-1beta) in rectal dialysate and plasma. Ulceration predominated in the distal colon in DSS-treated rats. There was no significant difference between the histological scores of the NF and NT-supplemented groups either at 7 or 12 d. MPO activity at 7 and 12 d was significantly higher in the NT-supplemented compared to NF group (7 d: 1013 +/- 172 vs. 409.9 +/- 103.2; 12 d: 471.9 +/- 112.4 vs. 223.6 +/- 21.6 units. min-1. g colon-1). IL-1beta concentration in rectal dialysate was significantly higher at 7 d in both groups compared to 0 and 4 d. At 12 d it continued to be significantly elevated in the NT-supplemented group and was greater than in the NT-free group. Our data on the proinflammatory cytokine, in conjunction with MPO activity, strongly suggest that NT supplementation aggravates the severity of DSS-induced colitis in rats.

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