| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
CUTTING EDGE |
-Producing CD4+ T Cells That Respond to Bacterial Antigens1
*
Laboratory of Immunology, I. Medical Clinic, University of Mainz, Mainz, Germany; and
Institute of Pathology, University of Mainz, Mainz, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
but not IL-4 upon activation with
CD3/CD28 or autologous bacterial Ags, consistent with a Th1-type
cell response. Furthermore, chronic colitis in STAT-4 transgenic mice
could be adoptively transferred to SCID mice by colonic and splenic
CD4+ T cells that were activated with Ags from autologous
bacterial flora. These data establish a critical molecular signaling
pathway involving STAT-4 for the pathogenesis of chronic intestinal
inflammation, and targeting of this pathway may be relevant for the
treatment of colitis in humans. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(IL-4), STAT-3 and STAT-4
are strongly activated by IL-12 1, 2 . IL-12 is a heterodimeric
cytokine produced mainly by activated follicular dendritic cells, B
cells, and macrophages 3, 4, 5 . Binding of IL-12 to the high-affinity
IL-12 receptor results in rapid activation of the Jak2 and Tyk2
signaling proteins and finally dimerization of STAT-4 and STAT-3
transcription factors 2 . In primary CD4+ T lymphocytes,
STAT-4 is essential for IL-12-dependent IFN- promoter activity
by binding to a specific recognition sequence in the IFN-
promoter, thus providing a molecular explanation for the role of STAT-4
in Th1 T cell differentiation 3 . The critical role of the
IL-12/STAT-4 pathway in driving Th1 T cell responses is further
underlined by the finding that both IL-12 p40 4 and STAT-4 5
knockout mice manifest impaired Th1/IFN- cytokine responses. Recently, various animal models of colitis caused by dysregulated T cell functions demonstrated the key role of Th1 T cells in the pathogenesis of chronic intestinal inflammation 6, 7, 8 . Because there is increasing evidence that IL-12/STAT-4-driven Th1 responses predominate in human Crohn’s disease 9 , we analyzed the effect of STAT-4 overexpression in a transgenic mouse model.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The murine STAT-4 cDNA 10 was cloned downstream of the CMV promoter into the pcDNA3.1 expression vector (Invitrogen, San Diego, CA) yielding the pcDNA3.1S4 vector. A 3.6-kp NruI/PvuII fragment of pcDNA3.1S4 containing the STAT-4 expression cassette was microinjected into pronuclei of fertilized eggs of FVB/NHSD mice. Mice were maintained under specific pathogen-free conditions in isolated cages.
Identification of transgenic mice
Transgene-positive founder mice were identified by Southern blots and PCR amplification of genomic DNA isolated from the tail using the QiaAmp Tissue Kit (Qiagen, Hilden, Germany). Southern blots were probed with a radiolabeled 1.3-kb BamHI/EcoRI fragment of pcDNA3.1S4.
Immunization of STAT-4 transgenic mice
For immunization mice were i.p. injected with 100 µg dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)3 (Calbiochem, Bad Soden, Germany) in CFA (Sigma, Deisenhofen, Germany) or alum. Control transgenic mice were injected with PBS.
Semiquantitative PCR for transgene-specific STAT-4 mRNA
Total RNA of spleen cells was prepared using Qiagen RNeasy columns (Qiagen). PCR was performed with a STAT-4-specific primer (5'-gctgaatgacggtgcaaacgg-3') and a second transgene-specific primer (5'-gacagtgggagtggcacctt-3') derived from sequences of the construct. PCR with ß-actin primers (5'-tcctgtggcatccatgaa-3' and 5'-cgcagctcagtaacagtc-3') was made as control. Cycling conditions were as follows: 94°C for 30 s, 57°C for 30 s, and 72°C for 75 s for 30 cycles.
Electrophoretic mobility shift assay (EMSA)
Cells were incubated for 20 min with 10 U/ml rIL-12 (obtained from Genzyme, Germany), and, subsequently, nuclear proteins were extracted. EMSA studies were made as described 3 . For supershift assays, polyclonal anti-STAT-4 (L-18; Santa Cruz Biotechnology, Heidelberg, Germany) Abs were used.
Immunohistochemistry and double-staining analysis
Frozen sections were fixed in 4% paraformaldehyde and incubated
overnight with the primary Ab (polyclonal rabbit anti-STAT-4 Ab,
C20; Santa Cruz; monoclonal anti-mouse IFN- and TNF; PharMingen,
San Diego, CA). Sections were then incubated with a biotinylated
secondary IgG Ab (Vector, Burlingame, CA) followed by incubation with
streptavidin-conjugated Cy2 (Dianova, Hamburg, Germany). A second cycle
of staining was made using anti-CD4 as primary Ab (rat
anti-mouse CD4; PharMingen) and streptavidin-conjugated Cy3. Three
mice per group were analyzed.
Histologic score
The degree of inflammation on microscopic cross-sections of the colon was graded semiquantitatively from 0 to 4 (0, no signs of inflammation; 1, very low level; 2, low level of leukocytic infiltration; 3, high level of leukocytic infiltration, thickening of the colon wall; 4, transmural inflammation, loss of goblet cells, thickening of the colon wall), as previously described 7 . Grading was done in a blinded fashion by the same observer on at least three colon cross-sections in each animal per group.
Isolation of spleen CD4+ lymphocytes and lamina propria mononuclear cells (LPMC)
Spleen CD4+ T cells (>95% purity) were isolated using immunomagnetic beads (Dynal, Oslo, Norway) with subsequent bead detachment 3 . CD4-enriched LPMC were isolated from resected large bowel specimens as described 7 .
Cell culture and ELISA for cytokine analysis
A total of 1 x 106 cells from three mice per group were cultured in 1 ml complete RPMI 1640 7 in triplicate wells. Cells were activated with precoated anti-CD3 mAb (10 µg/ml) and soluble anti-CD28 Abs (1 µg/ml; PharMingen). Supernatants were removed after 48 h and assayed for cytokine concentration by ELISA (PharMingen).
Preparation of bacterial Ags and APCs
Colonic bacterial Ags were prepared as described 11 . For generation of APC, 2 x 107 freshly isolated spleen cells were incubated overnight with 100 µg/ml bacterial sonicates and subsequently irradiated with 3000 rad. For proliferation assays, 5 x 104 cells in triplicate wells were incubated with 5 x 104 Ag-pulsed APC for 5 days in complete media. 0.25 µCi/well [3H]thymidine (DuPont, Les Ulis, France) were added for 18 h. The incorporation of [3H]thymidine was determined by ß scintillation counting.
Adoptive transfer
Splenic CD4+ T cells and LPMC from STAT-4 transgenic mice and control mice were isolated as described above and incubated over a period of 4 days with APC that were loaded with autologous bacterial Ags. Finally, 1 x 106 cells were i.p. injected in CB.17 SCID mice, IL-12 p40 knockout mice (4; kindly donated by Dr. E. Schmitt, Department of Immunology, University of Mainz, Mainz, Germany), or wild-type control mice.
Statistical analysis
Data were compared by the student’s t test for independent events (Statworks for Macintosh).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because previous studies suggested a key role for IL-12 in the
pathogenesis of colitis 7, 8 , we generated STAT-4 transgenic mice
using a vector containing the murine STAT-4 cDNA under control of a
human CMV-promoter (Fig. 1
a).
Southern blot analysis (not shown) of the offspring mice demonstrated
chromosomal integration of the construct in 12 mice, and two
independent founders (no. 6 and 15) were used for additional
experiments.
|
b). This finding was associated with increased STAT-4
protein levels in splenic T cells of immunized transgenic mice compared
with control mice (Fig. 1c). Severe transmural colitis in immunized STAT-4 transgenic mice
Within 7–14 days after i.p. injection of DNP-KLH plus CFA STAT-4
transgenic mice developed macroscopic and histologic signs of severe
transmural colitis (Table I), which was
observed as long as the animals were followed (3 mo). The use of alum
instead of CFA for immunization resulted in the development of a milder
colitis in STAT-4 transgenic mice compared with DNP-KLH plus CFA. In
contrast, CFA-treated STAT-4 transgenic mice and DNP-KLH plus
CFA-treated nontransgenic control mice failed to develop colitis (Table I). DNP-KLH-induced colitis was accompanied by diarrhea, weight loss,
and massive thickening of the bowel wall (Figs. 2, a and g).
Histopathological analysis of the ileum and large bowel of
DNP-KLH-treated animals showed severe mucosal inflammation in STAT-4
transgenic mice (Fig. 2, b–f; Table I). This inflammatory
process was characterized by destruction of the crypt architecture and
dense infiltrates of granulocytes, macrophages, and lymphocytes in the
lamina propria.
|
|
To characterize colitis in STAT-4 transgenic mice, we performed
immunohistochemical studies. It was found that STAT-4 colitic mice
showed dense infiltrates of CD4+ T cells in the colon
compared with DNP-KLH-treated control mice (Fig. 3). To further determine STAT-4 protein
levels in transgenic T cells, we performed double-staining analysis. We
observed that colonic CD4+ T cells from STAT-4 transgenic
mice express higher amounts of STAT-4 compared with control cells (Fig. 3).
|
Next, we analyzed cytokine production by spleen T cells from
STAT-4 transgenic mice. We observed that anti-CD3/CD28-activated T
cells from immunized STAT-4 transgenic mice produced on average more
TNF (7 ng/ml) and IFN- (32 ng/ml) than T cells from untreated
control mice (TNF, 2 ng/ml; IFN-, 13 ng/ml) and DNP-KLH-treated
control mice (TNF, 3 ng/ml; IFN-, 18 ng/ml). In contrast, there was
a low production of the Th2 cytokine IL-4 in both immunized STAT-4
transgenic mice (2 ng/ml) and immunized control mice (2 ng/ml) compared
with untreated control mice (26 ng/ml). Furthermore, no significant
differences in IL-10 production were found (immunized STAT-4 transgenic
mice, 7 ng/ml; immunized control mice, 5 ng/ml; unimmunized mice, 8
ng/ml), suggesting a predominant Th1 cytokine profile in immunized
STAT-4 transgenic mice.
In additional studies, we focused on the expression of TNF and IFN-
in situ. In these studies, CD4+ T lymphocytes were analyzed
by double-staining analysis using specific Abs to CD4, IFN-, and
TNF. As shown in Fig. 3, the number of IFN-- and TNF-expressing
CD4+ T lymphocytes was strikingly higher in the inflamed
colon of STAT-4 transgenic mice compared with control mice, suggesting
an activation of Th1 T cells in the lamina propria.
CD4+ T cells of STAT-4 transgenic mice with colitis
produce IFN- and proliferate in response to Ags of the autologous
bacterial flora
Previous studies suggested an important role of the intestinal
microflora in T cell-dependent enterocolitis 11, 13, 14 . Because
intestinal T cells are tolerant to autologous bacterial Ags but
reactive to heterologous Ags 11 , we analyzed if colitis in STAT-4
transgenic mice is associated with a loss of tolerance toward
autologous bacterial Ags. Accordingly, we stimulated lymphocytes from
STAT-4 transgenic mice and control mice with sonicates from autologous
and heterologous bacterial flora 11 . It was found that the latter
cells proliferated when stimulated with Ags from the heterologous flora
(DNP-KLH, 11,016 ± 1,129 CPM; untreated control mice, 12,489 ± 2,080
CPM), as previously described 11 . However, lymphocytes from STAT-4
transgenic mice proliferated even stronger (28,499 ± 3,558 CPM) when
stimulated with Ags from the autologous flora and produced increased
amounts of IFN- (STAT-4 flora, 2900 pg/ml vs. flora untreated mice,
0 pg/ml), suggesting increased reactivity to autologous bacterial Ags
in STAT-4 transgenic mice.
Adoptive transfer of STAT-4 colitis in SCID mice by CD4+ T lymphocytes that respond to bacterial Ags
Finally, we analyzed if colitis could be adoptively
transferred by LPMC and CD4+ T cells from immunized STAT-4
transgenic animals to SCID mice. Accordingly, purified splenic
CD4+ T cells and LPMC from wild-type and STAT-4 transgenic
animals were incubated with APC loaded with bacterial Ags from the
autologous flora and finally injected in SCID mice. Reconstitution of
SCID mice with STAT-4 transgenic spleen T cells (histologic score,
2.5 ± 0.9) or LPMC (score, 2.3 ± 0.8) that were activated
with Ags from the autologous flora led to severe transmural colitis
that was indistinguishable from that of DNP-KLH-treated STAT-4 mice
(Table I). In contrast, no severe colitis was found in SCID mice
reconstituted with T cells from DNP-KLH-treated nontransgenic control
mice (Table I). Furthermore, no severe colitis was seen in T
cell-reconstituted IL-12-knockout mice or wild-type control mice (Table I), possibly due to an intact or even activated counter-regulatory Th2
and Th3 cytokine response in the lamina propria of these mice.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because Abs to IL-12 have been shown to abrogate established
Th1-dependent colitis 7, 8 , we focused in the present study on the
functional role of the STAT-4 pathway by using a transgenic mouse
system. Interestingly, we could not detect transgenic STAT-4 mRNA in
the spleen, liver, and colon of several STAT-4 founder lines,
suggesting that the CMV promoter was silenced at least in these
tissues. However, activation of the immune system by systemic
administration of DNP-KLH resulted in expression of transgenic STAT-4
mRNA and increased STAT-4 protein levels in lamina propria T cells of
STAT-4 transgenic mice but not nontransgenic control mice.
Overexpression of STAT-4 in immunized mice was associated with
macroscopic and histologic signs of colitis that was characterized by a
transmural inflammation of T cells expressing high levels of nuclear
STAT-4. These data demonstrate a predominant pathogenic role of STAT-4
in chronic colitis that is further underlined by recent studies showing
the absence of colitis in CD45RBhigh T cell-reconstituted
SCID mice in which the STAT-4 protein had been inactivated by
homologous recombination 8 . The presence of activated nuclear STAT-4
in splenic and lamina propria T cells of STAT-4 transgenic mice
correlated with high expression of the proinflammatory cytokines TNF
and IFN-. Together with the decreased production of IL-4, these data
suggest an important pathogenic role of Th1-type T cells for onset and
maintenance of colitis.
Studies in germfree mice suggested an important role of the intestinal microflora in the pathogenesis of colitis 6, 13 . This is supported by studies showing that T cells from C3H/HejBir mice that respond to coecal bacterial Ags are capable to adoptively transfer colitis 14 . Interestingly, mucosal inflammation in STAT-4 transgenic mice was restricted to sites with the highest bacterial load (terminal ileum, colon), suggesting that mucosal Ags could play a role in the pathogenesis of colitis. In support of this hypothesis, we found that spleen cells of colitic STAT-4 transgenic mice proliferate to autologous bacterial Ags presented on APCs and consecutively produce proinflammatory Th1 cytokines. Finally, colitis in STAT-4 transgenic mice could be adoptively transferred to SCID mice by LPMC and splenic T cells that were activated via APC loaded with autologous bacterial Ags. These data directly indicate a key role of T cells that respond to luminal Ags in the pathogenesis of colitis in STAT-4 transgenic mice, and a similar mechanism has been recently suggested for the pathogenesis of Crohn’s disease in humans 15 .
In summary, the present data provide further insights into the pathogenic role of the STAT-4 signaling protein in the pathogenesis of colitis and define a critical molecular signaling pathway for the development of colonic inflammation. Taken together with recent data on the role of IL-12 and STAT-4 in human Crohn’s disease, these data suggest that activation of the IL-12/STAT-4 pathway is a key regulatory mechanism in the pathogenesis of chronic intestinal inflammation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Markus F. Neurath, Laboratory of Immunology, I. Medical Clinic, University Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail address:
3 Abbreviations used in this paper: DNP-KLH, dinitrophenyl-keyhole limpet hemocyanin; LPMC, lamina propria mononuclear cell; EMSA, electrophoretic mobility shift assay.
Received for publication September 17, 1998. Accepted for publication December 10, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and type 1 cytokine responses. Immunity 4:471.[Medline]
expression by T cells. J. Exp. Med. 187:1225.
B. J. Virol. 72:180.This article has been cited by other articles:
|
|
 |
|
  S Brand Crohn's disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn's disease Gut, August 1, 2009; 58(8): 1152 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. O'Malley, R. D. Eri, G. L. Stritesky, A. N. Mathur, H.-C. Chang, H. HogenEsch, M. Srinivasan, and M. H. Kaplan STAT4 Isoforms Differentially Regulate Th1 Cytokine Production and the Severity of Inflammatory Bowel Disease J. Immunol., October 1, 2008; 181(7): 5062 - 5070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Weigmann, H. A. Lehr, G. Yancopoulos, D. Valenzuela, A. Murphy, S. Stevens, J. Schmidt, P. R. Galle, S. Rose-John, and M. F. Neurath The transcription factor NFATc2 controls IL-6-dependent T cell activation in experimental colitis J. Exp. Med., September 1, 2008; 205(9): 2099 - 2110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Villarino, D. Artis, J. S. Bezbradica, O. Miller, C. J. M. Saris, S. Joyce, and C. A. Hunter IL-27R deficiency delays the onset of colitis and protects from helminth-induced pathology in a model of chronic IBD Int. Immunol., June 1, 2008; 20(6): 739 - 752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Caprioli, M. Sarra, R. Caruso, C. Stolfi, D. Fina, G. Sica, T. T. MacDonald, F. Pallone, and G. Monteleone Autocrine Regulation of IL-21 Production in Human T Lymphocytes J. Immunol., February 1, 2008; 180(3): 1800 - 1807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Hildner, P. Schirmacher, I. Atreya, M. Dittmayer, B. Bartsch, P. R. Galle, S. Wirtz, and M. F. Neurath Targeting of the Transcription Factor STAT4 by Antisense Phosphorothioate Oligonucleotides Suppresses Collagen-Induced Arthritis J. Immunol., March 15, 2007; 178(6): 3427 - 3436. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Becker, H. Dornhoff, C. Neufert, M. C. Fantini, S. Wirtz, S. Huebner, A. Nikolaev, H.-A. Lehr, A. J. Murphy, D. M. Valenzuela, et al. Cutting Edge: IL-23 Cross-Regulates IL-12 Production in T Cell-Dependent Experimental Colitis. J. Immunol., September 1, 2006; 177(5): 2760 - 2764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C Mueller and A J Macpherson Layers of mutualism with commensal bacteria protect us from intestinal inflammation Gut, February 1, 2006; 55(2): 276 - 284. [Full Text] [PDF]
|
|
|
|
 |
|
 G. Ramesh, X. Alvarez, J. T. Borda, P. P. Aye, A. A. Lackner, and K. Sestak Visualizing Cytokine-Secreting Cells In Situ in the Rhesus Macaque Model of Chronic Gut Inflammation Clin. Vaccine Immunol., January 1, 2005; 12(1): 192 - 197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fiorucci, J. L. Wallace, A. Mencarelli, E. Distrutti, G. Rizzo, S. Farneti, A. Morelli, J.-L. Tseng, B. Suramanyam, W. J. Guilford, et al. A {beta}-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction PNAS, November 2, 2004; 101(44): 15736 - 15741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Weigmann, A. Nemetz, C. Becker, J. Schmidt, D. Strand, H. A. Lehr, P. R. Galle, I.-C. Ho, and M. F. Neurath A Critical Regulatory Role of Leucin Zipper Transcription Factor c-Maf in Th1-Mediated Experimental Colitis J. Immunol., September 1, 2004; 173(5): 3446 - 3455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sestak, C. K. Merritt, J. Borda, E. Saylor, S. R. Schwamberger, F. Cogswell, E. S. Didier, P. J. Didier, G. Plauche, R. P. Bohm, et al. Infectious Agent and Immune Response Characteristics of Chronic Enterocolitis in Captive Rhesus Macaques Infect. Immun., July 1, 2003; 71(7): 4079 - 4086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Marhaba, M. Bourouba, and M. Zoller CD44v7 interferes with activation-induced cell death by up-regulation of anti-apoptotic gene expression J. Leukoc. Biol., July 1, 2003; 74(1): 135 - 148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Lovato, C. Brender, J. Agnholt, J. Kelsen, K. Kaltoft, A. Svejgaard, K. W. Eriksen, A. Woetmann, and N. Odum Constitutive STAT3 Activation in Intestinal T Cells from Patients with Crohn's Disease J. Biol. Chem., May 2, 2003; 278(19): 16777 - 16781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I Monteleone, P Vavassori, L Biancone, G Monteleone, and F Pallone Immunoregulation in the gut: success and failures in human disease Gut, May 1, 2002; 50(90003): iii60 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M-N Kweon, I Takahashi, M Yamamoto, M H Jang, N Suenobu, and H Kiyono Development of antigen induced colitis in SCID mice reconstituted with spleen derived memory type CD4+ CD45RB+ T cells Gut, March 1, 2002; 50(3): 299 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Hendrickson, R. Gokhale, and J. H. Cho Clinical Aspects and Pathophysiology of Inflammatory Bowel Disease Clin. Microbiol. Rev., January 1, 2002; 15(1): 79 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wirtz, C. Becker, R. Blumberg, P. R. Galle, and M. F. Neurath Treatment of T Cell-Dependent Experimental Colitis in SCID Mice by Local Administration of an Adenovirus Expressing IL-18 Antisense mRNA J. Immunol., January 1, 2002; 168(1): 411 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Finotto, G. T. De Sanctis, H. A. Lehr, U. Herz, M. Buerke, M. Schipp, B. Bartsch, R. Atreya, E. Schmitt, P. R. Galle, et al. Treatment of Allergic Airway Inflammation and Hyperresponsiveness by Antisense-Induced Local Blockade of Gata-3 Expression J. Exp. Med., June 4, 2001; 193(11): 1247 - 1260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Parrello, G. Monteleone, S. Cucchiara, I. Monteleone, L. Sebkova, P. Doldo, F. Luzza, and F. Pallone Up-Regulation of the IL-12 Receptor {beta}2 Chain in Crohn's Disease J. Immunol., December 15, 2000; 165(12): 7234 - 7239. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
