 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
*
Department of Nephrology, University of Heidelberg, Heidelberg,
Germany
Department of Pathology, University of Heidelberg, Heidelberg,
Germany
Department of Nephrology, University of Hannover, Hannover,
Germany.
Correspondence to Dr. Jürgen Wagner, Department of Nephrology, University Hospital, University of Heidelberg, Bergheimerstrasse 56a, D-69115 Heidelberg, Germany. Phone: +49 6221 91120; Fax: +49 6221 16 24 76; E-mail: juergen_wagner{at}med.uni-heidelberg.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the search for substances that interfere with this proliferative process, retinoids, i.e., natural or synthetic derivatives of vitamin A, are of great interest because they are known to exert strong antiproliferative effects in most cell types, such as myocytes, vascular smooth muscle cells, and mesangial cells (5,6,7). Additionally, they have an anti-inflammatory action and modulate extracellular matrix synthesis in a complex and tissue-specific manner (8). These processes are also relevant for kidney disease.
Retinoic acids (RA) act via RA receptors (RAR) 
, ß, and 
and retinoid X receptors (RXR) , ß, and
(9,
10). The antiproliferative
effects of retinoids are attributable to modulation of gene transcription by
these compounds
(11,12,13).
RA and synthetic retinoids demonstrate potent anti—activator protein-1
(AP-1) activity and have been proposed as substances that downregulate
AP-1-regulated genes. RA interfere with other proinflammatory pathways as well
as nuclear factor-
B and creb-binding protein/P300, which integrate a
number of cell regulatory and signaling pathways
(14,15,16).
A role for retinoids in kidney differentiation has been demonstrated in
double-knockout mice deficient for RAR-/RXR-. In tissue culture,
retinoids influence glomerular number and tubular differentiation
(17,18,19).
Little is known regarding the effects of retinoids on kidneys of mature
animals or human subjects (20,
21). The antiproliferative
effects of retinoids prompted us to examine whether retinoids reduce mesangial
cell proliferation and thus limit glomerular injury in a rat model of
mesangioproliferative glomerulonephritis. We studied the effects of
all-trans-RA, a prototypic natural retinoid, and of isotretinoin
(13-cis-RA), a second generation retinoid that is far less toxic and
is used in human therapeutic regimens. Two protocols were used. In the first
series, treatment with all-trans-RA was initiated before injection of
the anti-Thy1.1 antibody (pretreatment). In the second series, treatment of
rats with all-trans-RA or isotretinoin was started on the third day
after antibody injection (posttreatment).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the second set of experiments, treatment with daily subcutaneous injections of 10 mg/kg body wt all-trans-RA or vehicle began 2 d after intravenous injection of OX-7 or PBS (control) (posttreatment). This set of experiments included two additional experimental groups, which were treated with daily subcutaneous injections of 40 mg/kg body wt isotretinoin (13-cis-RA) (Hoffmann LaRoche, Basel, Switzerland) dissolved in arachis oil and 5% DMSO, as described above. One of those groups received the OX-7 antibody and the other received PBS (control) 2 d before treatment with retinoids. In the second set of experiments, renal biopsies were performed on day 4 after antibody injection and animals were euthanized on day 8 after induction of nephritis.
BP was determined on days -3 and +8 by tail-cuff plethysmography, with light ether anesthesia. For euthanasia, animals were given intramuscular injections of xylazine (5 mg/kg body wt; Bayer Vital, Leverkusen, Germany) and ketamine (10%, 100 mg/kg body wt; WDT, Garbsen, Germany). Rats were perfused with saline solution containing 0.5 g/L procaine hydrochloride, at a defined pressure of 110 mmHg, by retrograde insertion of a cannula into the abdominal aorta (23). The inferior vena cava was incised to drain blood. Creatinine clearance was calculated after enzymatic determination (using a creatinine kit; Hoffmann LaRoche) of serum and urinary (from 24-h urine samples) creatinine levels, with a Hitachi autoanalyzer (Frankfurt, Germany).
Signs of retinoid toxicity were apparent after treatment with all-trans-RA and included cheilitis and some hair loss. Animals treated with all-trans-RA lost approximately 8% of their body weight (217 ± 4.1 g) compared with the anti-Thy1.1/vehicle-treated group (233 ± 3.1 g), whereas no weight loss was observed for the anti-Thy1.1/isotretinoin-treated group (233 ± 4.2 g).
Renal Morphologic Studies
Tissue for light microscopy was fixed in 10% buffered formalin and embedded
in paraffin. Sections (4 µm) were stained with the periodic acid-Schiff
(PAS) reagent and counterstained with hematoxylin. In the PAS-stained
sections, the total number of nuclei in each glomerular cross section was
determined, and mesangiolysis was graded semiquantitatively on a scale from 0
to 3+, as described (24). For
all morphologic determinations, the investigator was blinded with respect to
experimental groups. Accuracy was determined by a second investigator for some
parameters. A minimum of 30 cortical glomeruli with diameters of at least 100
µm were examined in each biopsy specimen.
Capillary Occlusion Scores In PAS-stained sections, the extent of occlusion of the capillary tuft area was determined using a semiquantitative scoring system (0, little or no occlusion; 1 +, up to 25% of the total capillary tuft area occluded; 2+, 25 to 50% occluded; 3+, 50 to 75% occluded; 4+, > 75% of the capillary tuft area occluded in at least 30 cortical glomeruli).
Free Bowman Space Morphometric analysis was performed using PAS-stained sections. At least 100 glomeruli were analyzed for each animal. For area measurements, a semiautomatic video image-analyzing system (Videoplan; Kontron Co., Eching, Germany) was used (23). At a primary magnification of x160, the mean area of glomerular profiles was measured. The area included within the Bowman capsule was determined and the total capillary tuft area was subtracted, to calculate the free Bowman space. No significant difference was observed in the area including the Bowman capsule among the different groups.
Total Glomerular Cell Counts Total glomerular cell counts were determined in at least 30 cortical glomeruli with diameters of at least 100 µm for each kidney, in PAS-stained sections (25). Visible glomerular mitoses (anaphase and metaphase) were counted in 100 glomeruli for each kidney, in PAS-stained sections.
Immunohistochemistry
Saline-perfused slices (4 µm) of renal tissue obtained from comparable
renal areas in all rats were fixed in methyl Carnoy's solution and processed
using the direct or indirect immunoperoxidase technique, as described
previously (22). The primary
antibodies used included those described previously
(22,
25): 1A4 (an antibody against
-smooth muscle actin [-SMA]; Dako, Glostrup, Denmark)
(26), 19A2 (a murine IgM
monoclonal antibody against human proliferating cell nuclear antigen [PCNA];
Coulter, Hialeah, FL), and PGF-007 (a murine monoclonal antibody against the
human platelet-derived growth factor [PDGF] B-chain; Mochida Pharmaceutical,
Tokyo, Japan).
For all biopsies, negative controls involved substitution of the primary
antibody with equivalent concentrations of an irrelevant murine monoclonal
antibody or normal rabbit IgG. For each biopsy, more than 30 cross sections of
consecutive cortical glomeruli with diameters of at least 100 µm were
evaluated, in a blinded manner. Mean values of the number of proliferating
(PCNA-positive) cells per biopsy were calculated. For evaluation of
immunoperoxidase staining for -SMA, each glomerulus was graded
semiquantitatively, as described
(25), and the mean score per
biopsy was calculated. The scores reflect changes in the extent but not the
intensity of mesangial matrix staining (0, diffuse weak staining; 1+, up to
25% of the glomerular tuft exhibiting focally increased staining; 2+, 25 to
50% of the tuft exhibiting staining; 3+, 50 to 75% of the tuft exhibiting
staining; 4+, > 75% of the glomerular tuft exhibiting strong staining). For
double-staining of proliferating cells with anti-PCNA and anti--SMA
antibodies (see above), we used the Super Sensitive ISH Detection System
(Biogenex Laboratories, San Ramon, CA), according to the recommendations of
the manufacturer, with 3,3'-diaminobenzidine chromogen for PCNA and
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate for
-SMA. Fibrin deposition was stained according to the method of Masson
(27).
Albuminuria
For determination of albumin levels in urine, rats were placed in metabolic
cages and urine was collected for 24 h. Albuminuria in rats was determined
essentially as described by Magnotti et al.
(28), on a 96-well
enzyme-linked immunosorbent assay plate, using a peroxidase-conjugated
anti-rat albumin antibody (ICN-Biomedical, Eschwege, Germany). All
measurements were performed in quadruplicate.
Statistical Analyses
All values are expressed as mean ± SEM, unless stated otherwise.
Statistical significance (defined as P < 0.05) was evaluated using
the nonparametric Mann-Whitney test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
Effects of RA on Renal Histologic Features
Figure 4 depicts typical
PAS-stained sections of glomeruli from animals with anti-Thy1.1-induced
glomerulonephritis, after treatment with vehicle
(Figure 4A) or
all-trans-RA (Figure
4B). Note the reduction in swelling of the glomerular tuft in the
all-trans-RA treated rat. The capillary occlusion score was
significantly higher for vehicle-treated rats with anti-Thy1.1-induced
glomerulonephritis compared with controls, but was significantly lower with
all-trans-RA pretreatment (P < 0.001)
(Figure 5A). The number of
totally occluded glomeruli was also significantly less in
all-trans-RA-treated nephritic rats (11.2 ± 1 out of 50
glomeruli in the anti-Thy1.1/vehicle-treated group versus 1.2
± 0.2 in the anti-Thy1.1/RA-treated group, P < 0.004). In
these animals, the free Bowman space was completely preserved (i.e.,
glomerular tuft swelling was prevented) by pretreatment with
all-trans-RA (Figure
5B).
|
|
The number of cells per glomerular cross section was significantly increased in vehicle-treated glomerulonephritic rats compared with control rats. Pretreatment with all-trans-RA did not affect glomerular cell counts in control rats but significantly reduced glomerular cell counts in rats with anti-Thy1.1-induced glomerulonephritis, compared with vehicle-treated nephritic rats (P < 0.01) (Figure 6A). Similarly, posttreatment with all-trans-RA and isotretinoin decreased glomerular cell numbers (control/vehicle, 69 ± 1.9; anti-Thy1.1/vehicle, 106 ± 2.7; anti-Thy1.1/isotretinoin, 95 ± 2.1; anti-Thy1.1/RA, 92 ± 2.2; P < 0.02, compared with the anti-Thy1.1/vehicle-treated group). Mesangiolysis was examined in renal biopsies obtained on day 4 in the posttreatment experiments. On day 4, we did not observe any difference in the extent of mesangiolysis in the nephritic rats treated with either vehicle, isotretinoin, or all-trans-RA (anti-Thy1.1/vehicle, 1.85 ± 0.09; anti-Thy1.1/isotretinoin, 1.8 ± 0.1; anti-Thy1.1/RA, 1.81 ± 0.13; NS). Mesangiolysis was barely detectable in anti-Thy1.1/vehicle-treated rats on day 8.
|
Effects of RA on Glomerular Cell Proliferation
The number of visible mitoses in glomerular cells was lower in rats
pretreated with all-trans-RA compared with vehicle-treated rats with
anti-Thy1.1-induced glomerulonephritis (P < 0.001)
(Figure 6B). Figure 4 depicts typical
glomerular staining for PCNA-positive cells in rats with anti-Thy1.1-induced
nephritis, without (Panel C) or with (Panel D) all-trans-RA. The
number of PCNA-positive cells was markedly increased in nephritic rats
compared with control rats, but was significantly less in
all-trans-RA-treated rats compared with vehicle-injected rats
(P < 0.001) (Figure
7). The PCNA-positive glomerular cell number was low in control
rats without glomerulonephritis.
|
The experiments involving posttreatment with all-trans-RA or
isotretinoin revealed significantly reduced staining of glomerular cells for
-SMA compared with untreated glomerulonephritic rats (-SMA
scores: anti-Thy1.1/vehicle, 2.6 ± 0.1; anti-Thy1.1/isotretinoin, 2.0
± 0.3; anti-Thy1.1/RA, 1.83 ± 0.2; both P < 0.02
versus anti-Thy1.1/vehicle; control/vehicle 0.2 ± 0.1).
Double-staining for PCNA and -SMA revealed colocalization of these
markers primarily in mesangial cells
(Figure 4, E and F). In the
posttreatment experiments, glomerular staining for PDGF-B revealed
significantly reduced staining in the glomeruli of anti-Thy1.1-treated
nephritic animals treated with isotretinoin or all-trans-RA, compared
with vehicle-treated rats (Figure
8).
|
Effects of RA on Monocytes/Macrophages and Glomerular Fibrin
Deposition
The number of glomerular macrophages/monocytes was markedly higher in
anti-Thy1.1/vehicle-treated rats than in control animals
(Figure 9A). Isotretinoin
significantly reduced the number of macrophages compared with
anti-Thy1.1-treated nephritic rats, whereas reduction in the presence of
all-trans-RA did not reach statistical significance. Deposition of
glomerular fibrin was enhanced in anti-Thy1.1-treated nephritic rats and was
significantly less in anti-Thy1.1/RA-treated rats (P < 0.001)
(Figure 9B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The preservation of glomerular structure by retinoids may be attributable to their antiproliferative action, which is a main characteristic of these compounds. In addition to carcinoma or
HeLa cells, antiproliferative effects of retinoids were observed for all cell types investigated, e.g., vascular smooth muscle cells, cardiac myocytes, and mesangial cells stimulated with fetal calf serum (5,6,7,13). Inhibition of proliferation is presumably attributable to inhibition of AP-1 transactivation (11,12) as a result of direct protein-protein interactions of retinoid receptors with the AP-1 complex, downregulation of c-Fos and Jun-1, or other mechanisms (11). This effect of retinoids has been examined only in cell culture experiments, but it may explain the inhibition of genes that are involved in glomerular damage and act via AP-1, such as angiotensin II, PDGF, and the endothelins (5,29,30). Angiotensin II-induced growth promotion in vascular smooth muscle cells can be blocked by all-trans-RA (29). Similarly, retinoids block the cell hypertrophy induced by endothelin-1 in embryonic cardiac myocytes (5). PDGF-B was shown to be inhibited by retinoids during mouse embryogenesis (30). In this study, glomerular staining for PDGF-B was less intense after treatment with either all-trans-RA or isotretinoin. In this model, PDGF may serve as a marker of proliferation. This notion is in agreement with the finding that fewer cells express PCNA in the presence of retinoids. It is unlikely that the antiproliferative action of retinoids is mediated only by inhibition of the AP-1 complex. These compounds influence protooncogenes, such as c-myc and the H-ras pathway (31), and compete for creb-binding protein/P-300, an integrator complex that integrates a number of signaling pathways. The antiproliferative function of retinoids has led to their use in the treatment of cancer and hyperplastic skin diseases (32,33,34).
All-trans-RA is used for human patients for the treatment of leukemais or prostate or metastatic lung disease, but even low doses may cause vitamin A-like toxicity. Isotretinoin, a second-generation retinoid, is much less toxic and is used in human patients for the treatment of severe acne (35). The purpose of our study was to establish whether retinoids can influence renal damage in nephritis. We therefore selected high doses of all-trans-RA and isotretinoin, which exceeded dose recommendations for human subjects (rat toxicity data were kindly provided by Hoffmann LaRoche, courtesy of Dr. M. Klaus, in a personal communication). We observed weight loss (-7%) for all-trans-RA-treated rats, with some hair loss, cheilitis, and keratitis, but animals treated with isotretinoin demonstrated no signs of toxicity.
All-trans-RA is known to interact specifically with the RAR. However, in tissues and organs, natural retinoids may isomerize into different retinoid forms, which may interact with RAR or RXR in a tissue-specific manner. Therefore, the experiments described above do not permit conclusions regarding which receptors in the kidney are responsible for the observed retinoid effects. RAR were demonstrated to mediate retinoid- and vitamin A-specific effects but may be differentially expressed during development (9, 11). In contrast to RAR, RXR form heterodimers with other members of the steroid receptor superfamily, enabling cross-talk with the vitamin D receptor, peroxisome-proliferative activator receptor, thyroid receptor, or other receptors (36, 37). Little is known regarding the expression of these receptors in the kidney. In renal failure, impairment of the interaction between vitamin D and RXR, because of downregulation of RXR, was observed (37). During ontogeny, double-knockout mice deficient for RAR isotypes show kidney agenesis or aplasia (17). Vitamin A has teratogenetic effects involving renal development (38). Retinoids control tubular and glomerular development in metanephros cultures (39). These observations indicate that retinoids are closely involved in renal development. Our findings support the notion that these vitamin A derivatives may also play a role in kidney disease in adults. The anti-Thy1.1-induced nephritis model has frequently been used to test potential therapeutic candidates for the treatment of this disease (22,25,40), although no specific treatment modality has yet been developed for the treatment of human IgA nephritis. Much work needs to be performed before the clinical application of retinoids for the treatment of human renal disease might be warranted. However, the pharmacologic features of retinoids have been well described, specific retinoid receptor agonists and antagonists are available, and clinical experience with retinoid treatment of human diseases has already been accumulated. These pharmacologic tools may facilitate the detailed analysis of retinoid effects in renal disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
involves
transcription factor NF-B. J Biol Chem271
: 26954-26961,1996This article has been cited by other articles:
 |
|
 |
|
  T.-C. Lu, Z. Wang, X. Feng, P. Chuang, W. Fang, Y. Chen, S. Neves, A. Maayan, H. Xiong, Y. Liu, et al. Retinoic Acid Utilizes CREB and USF1 in a Transcriptional Feed-Forward Loop in Order To Stimulate MKP1 Expression in Human Immunodeficiency Virus-Infected Podocytes Mol. Cell. Biol., September 15, 2008; 28(18): 5785 - 5794. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang, H. Meng, Z.-H. Li, Z. Shu, X. Ma, and B.-X. Zhang Regulation of STIM1, store-operated Ca2+ influx, and nitric oxide generation by retinoic acid in rat mesangial cells Am J Physiol Renal Physiol, March 1, 2007; 292(3): F1054 - F1064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. He, T.-C. Lu, M. Fleet, M. Sunamoto, M. Husain, W. Fang, S. Neves, Y. Chen, S. Shankland, R. Iyengar, et al. Retinoic Acid Inhibits HIV-1-Induced Podocyte Proliferation through the cAMP Pathway J. Am. Soc. Nephrol., January 1, 2007; 18(1): 93 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xing, J. Ding, Q. Fan, and N. Guan Diversities of Podocyte Molecular Changes Induced by Different Antiproteinuria Drugs. Experimental Biology and Medicine, May 1, 2006; 231(5): 585 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Wen, Y. Li, K. Hu, C. Dai, and Y. Liu Hepatocyte Growth Factor Receptor Signaling Mediates the Anti-Fibrotic Action of 9-cis-Retinoic Acid in Glomerular Mesangial Cells Am. J. Pathol., October 1, 2005; 167(4): 947 - 957. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yamauchi, A. Ishibashi, K. Shikata, N. Tokuhara, K.-i. Seino, S. Kobayashi, and M. Nagai Effect of E6060 [4-{5-[7-Fluoro-4-(trifluoromethyl)benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic Acid], a Novel Subtype-Selective Retinoid, on Lupus-Like Nephritis in Female (NZBxNZW)F1 Mice J. Pharmacol. Exp. Ther., March 1, 2005; 312(3): 938 - 944. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Perez, M. Ramirez-Ramos, C. Calleja, D. Martin, M. C. Namorado, G. Sierra, M. E. Ramirez-Ramos, R. Paniagua, Y. Sanchez, L. Arreola, et al. Beneficial effect of retinoic acid on the outcome of experimental acute renal failure Nephrol. Dial. Transplant., October 1, 2004; 19(10): 2464 - 2471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Liebler, B. Uberschar, H. Kubert, S. Brems, A. Schnitger, M. Tsukada, C. C. Zouboulis, E. Ritz, and J. Wagner The renal retinoid system: time-dependent activation in experimental glomerulonephritis Am J Physiol Renal Physiol, March 1, 2004; 286(3): F458 - F465. [Abstract] [Full Text]
|
|
|
|
 |
|
 K. Kinoshita, B.-S. Yoo, Y. Nozaki, M. Sugiyama, S. Ikoma, M. Ohno, M. Funauchi, and A. Kanamaru Retinoic Acid Reduces Autoimmune Renal Injury and Increases Survival in NZB/W F1 Mice J. Immunol., June 1, 2003; 170(11): 5793 - 5798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Suzuki, T. Ito, E. Imai, M. Yamato, H. Iwatani, H. Kawachi, and M. Hori Retinoids Regulate the Repairing Process of the Podocytes in Puromycin Aminonucleoside-induced Nephrotic Rats J. Am. Soc. Nephrol., April 1, 2003; 14(4): 981 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. MORATH, C. DECHOW, I. LEHRKE, V. HAXSEN, R. WALDHERR, J. FLOEGE, E. RITZ, and J. WAGNER Effects of Retinoids on the TGF-{beta} System and Extracellular Matrix in Experimental Glomerulonephritis J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2300 - 2309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. LUCIO-CAZANA, K. NAKAYAMA, Q. XU, T. KONTA, V. MORENO-MANZANO, A. FURUSU, and M. KITAMURA Suppression of Constitutive but Not IL-1{beta}-Inducible Expression of Monocyte Chemoattractant Protein-1 in Mesangial Cells by Retinoic Acids: Intervention in the Activator Protein-1 Pathway J. Am. Soc. Nephrol., April 1, 2001; 12(4): 688 - 694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Haxsen, S. Adam-Stitah, E. Ritz, and J. Wagner Retinoids Inhibit the Actions of Angiotensin II on Vascular Smooth Muscle Cells Circ. Res., March 30, 2001; 88(6): 637 - 644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wagner Potential role of retinoids in the therapy of renal disease Nephrol. Dial. Transplant., March 1, 2001; 16(3): 441 - 444. [Full Text] [PDF]
|
|
|
|
|
|
I. Lehrke, M. Schaier, K. Schade, C. Morath, R. Waldherr, E. Ritz, and J. Wagner Retinoid receptor-specific agonists alleviate experimental glomerulonephritis Am J Physiol Renal Physiol, April 1, 2002; 282(4): F741 - F751. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
