| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Divisions of
*
Rheumatology and
Renal-Electrolyte and Hypertension, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57B6 cGVH model. CD4KO
B6 mice injected with allogeneic bm12 spleen cells (bm12CD4KO group)
showed no evidence of cGVH disease. They made no detectable
autoantibodies, including anti-chromatin, anti-dsDNA,
anti-ssDNA, and rheumatoid factor. They survived at least 20 wks
after induction of cGVH disease; and they did not develop nephritis,
based on the absence of detectable levels of proteinuria and normal
renal histology at the time of sacrifice. By contrast, CD8KO B6 mice
(bm12CD8KO group) and normal B6 mice (bm12B6 group) injected with
bm12 spleen cells generally showed similar levels of mortality,
nephritis, and autoantibodies, although the autoantibody titers
declined somewhat after week 8 in the bm12CD8KO group. Control
groups of recipients injected with B6 spleen cells showed no induction
of autoantibodies. A surprising finding, however, was that the
B6CD8KO group developed severe histologic glomerulonephritis in the
absence of autoantibodies and with decreased immune deposits. These
results indicate that endogenous (host) CD4+ T cells play
an essential role in the cGVH autoimmune
syndrome. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(DBA/2 x
C57BL/6)F1 and other parentF1 strain
combinations used by other investigators (reviewed in Refs. 6 and 7).
In the DBA/2(DBA/2 x C57BL/6)F1 model, both MHC
class I and class II differences are present between donor and
recipient, and the development of cGVH disease depends on a relative
inactivity of the donor CD8+ T cells (6). Accordingly, this
system can be driven toward an acute GVH by the in vivo administration
of rIL-12 (8) or anti-OX40 Ab (9), or by increasing the percentage
of donor CD8+ T cells (10). Our working hypothesis for the cellular mechanism of this autoimmune syndrome has been that the alloreactive donor T cells recognize the recipient B cells’ MHC class II molecules together with some peptides that may or may not be derived from the autoantigens against which the B cells react. This allogeneic effect then delivers an abnormal T cell help signal to the B cells and drives them to produce inappropriate specificities, i.e., autoantibodies. Such a model would not postulate any essential role for the host’s endogenous T cells. Nevertheless, several lines of evidence have suggested that recipient T cells might be involved in the cGVH autoimmune responses. Rolink et al. (11) reported that adult-thymectomized, irradiated, bone marrow-reconstituted recipients of alloreactive T cells had a more severe cGVH syndrome than intact recipients. On the other hand, repeated attempts in our laboratory to induce cGVH in athymic (nu/nu) B6 recipients by the transfer of varying numbers of bm12 spleen cells have not led to the production of autoantibodies (our unpublished data). Finally, Gonzalez et al. (12) observed that the ability of B cells from F1 hybrid mice to respond to allogeneic help from parental T cells in vitro depended on the presence of CD4+ T cells in the B cell donor. Collectively, these studies suggest that the cellular interactions that induce the production of autoantibodies in the cGVH may require recipient T cells.
To address this issue, we used CD4 knockout (KO) and CD8KO B6 mice as
recipients in our cGVH model (13, 14). In these strains, the CD4 or
CD8
genes have been inactivated by homologous recombination. We
found that allogeneic splenocyte transfer did not induce a cGVH disease
in CD4KO mice, as determined by mortality, autoantibody production, and
renal disease. CD8KO recipients, on the other hand, produced somewhat
lower levels of autoantibodies in the late stage of cGVH disease than
normal B6 recipients, while they showed comparable severity of renal
disease. These results indicate that host (endogenous) T cells,
especially host CD4+ T cells, play very important roles in
the cGVH autoimmune responses.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6-Cd4tm1Mak (CD4KO), C57BL/6J (B6), and B6.C-H2bm12/KhEg (bm12) mice, originally obtained from The Jackson Laboratory (Bar Harbor, ME), and C57BL/6-Cd8atm1Mak (CD8KO) mice, originally obtained from Dr. Tak W. Mak (University of Toronto, Canada), were bred and maintained in our mouse colony at the University of Pennsylvania Medical Center. Strains B6 and bm12 differ only by three amino acids in the ß-chain of the I-A molecule. Recipient and donor mice were sex- and age-matched within each independent experiment.
Experimental cGVH disease protocol
cGVH disease was induced as previously described (1). Briefly, recipient mice between 2–5 mo of age were injected (i.p.) with single cell suspensions of 1 x 108 donor splenocytes, prepared by pressing donor spleens through a wire mesh screen in HBSS. Blood samples were obtained from experimental mice at the induction of cGVH disease and at 2- to 4-wk intervals thereafter. Sera were stored at -20°C for later analysis.
Detection of autoantibodies
The expression of autoantibodies was assessed by ELISA, as
previously described (4). Autoantigens (see below) were diluted in
borate-buffered saline (BBS), added to polyvinyl microtiter plates
(Dynatech Laboratories, Alexandria, VA), and incubated 4 h at room
temperature or overnight at 4°C. The plates were washed with BBS and
blocked with BBS supplemented with Tween and BSA (BBT:BBS, 0.4% Tween
80, 0.5% BSA, and 0.1% NaN3) for 1 h at room
temperature. Serum samples, diluted 1/500 (1:1000 for RF) in BBT, were
added in duplicate and incubated 5 h at room temperature or
overnight at 4°C. For reference, a standard serum from
B6/lpr mouse with high titer of autoantibodies was tested at
13 different dilutions from 1:250 to 1:1,024,000. The plates were
washed with BBS, and biotinylated rat anti-mouse Ig
(
-chain-specific) or biotinylated F(ab')2 rat
anti-mouse IgM (Bet-2, for detecting rheumatoid factor (RF)),
diluted 1/2000 in BBT, was added and incubated 4 h at room
temperature. The plates were washed and incubated for 1 h at room
temperature with avidin-alkaline phosphatase (Zymed Laboratories, South
San Francisco, CA). The plates were washed again, and paranitrophenyl
phosphate substrate (Sigma, St. Louis, MO), 1 mg/ml in 0.01 M
diethanolamine, pH 9.8, was added. The plates were read at various time
points with an automated ELISA reader (Dynatech). Autoantibody results
from individual ELISAs were standardized against the reference serum,
and the result for each sample was defined as an equivalent dilution
factor (EDF = (the dilution of the reference B6/lpr
serum that gives an assay OD equal to that of the sample serum) x
106) (15).
Autoantigens
1) Chromatin, purified from chicken erythrocyte nuclei, was used at 5 µg/ml. 2) dsDNA from calf thymus (Sigma) was used at 3 µg/ml. 3) ssDNA, made from dsDNA by heating dsDNA at 97°C for 10 min and cooling on ice quickly, was used at 2.5 µg/ml. 4) IgG2bb (clone CBPC101) was used at 3 µg/ml for IgM RF autoantigen.
Total IgG and Total IgM
Total IgG and total IgM were also assessed by ELISA as described above. Instead of coating plates with autoantigens, goat anti-mouse Fab at 3 µg/ml for total IgG or goat anti-mouse IgM at 4 µg/ml for total IgM, were coated onto plates as the first step of the ELISA. The biotinylated Abs were goat anti-mouse pFc' for total IgG and Bet-2 F(ab')2 for total IgM. Mouse IgG (clone HB63) and mouse IgM (clone CBPC 112) were used as standards in these assays.
Evaluation of nephritis
Urine protein concentrations were detected at 2- to 4-wk intervals using Uristix reagent strips (Miles Laboratories, Elkhart, IN). The presence and severity of nephritis was determined by hematoxylin and eosin light and immunofluorescence microscopy, as previously described (16). Briefly, for light microscopy, the severity of glomerular, interstitial, and vascular lesions was determined independently by blinded grading by one of us (M.M.) on a scale of 0 to 4+. Similarly, the presence of glomerular, tubular basement membrane and vascular immune deposits were judged independently. Multiple sections at a minimum of two different levels were observed. Each section typically involved evaluation of over 50 glomeruli, more than 25 blood vessels, and the interstitium contained within two to three longitudinal sections of kidney.
Statistical and data analyses
The autoantibodies and proteinuria data presented are from a single CD4KO experiment or from two pooled CD8KO experiments, which individually showed similar results. Two subsequent experiments with the CD4KO recipients and one with the CD8KO recipients again gave comparable results. Statistical analysis of data was performed according to the Student t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cGVH disease was established in unirradiated recipient mice (Table I
) by i.p. injections of a single dose of
1 x 108 age/sex-matched donor cells. Mice were
followed for survival and for periodic determination of urinary protein
and collection of serum. As shown in Fig. 1, the bm12CD8KO group and bm12B6
groups showed similar mortality rates, reaching 50–60% at week 20
after the induction of cGVH disease. By contrast, 10/11 mice in the
B6CD8KO groups and all mice in B6CD4KO and bm12CD4KO groups
survived to week 24. Additional control groups of B6B6
(n = 10), CD4KOCD4KO (n = 6), and
CD8KOCD8KO (n = 8) also showed no mortality by week
20 (data not shown). The difference in mortality rates
between bm12B6 and bm12CD4KO groups was highly
significant (p < 0.01).
|
|
The levels and specificities of autoantibody production during
cGVH disease were assessed by ELISA. Surprisingly, the bm12CD4KO
group showed no autoantibody titers different from the negative control
group (B6CD4KO), except for a modest, transient elevation of
anti-chromatin and RF at week 2, and anti-dsDNA at week 4 (Fig. 2A). As expected, the
concomitant bm12B6 positive control group showed substantial
elevations of autoantibody titers of anti-chromatin, RF,
anti-ssDNA, anti-dsDNA at all time points tested after week 0.
The bm12CD8KO group also had significant levels of
anti-chromatin, RF, anti-ssDNA, anti-dsDNA compared with
the negative control (B6CD8KO group) at all time points except week
0 (p < 0.01). However, the concomitant
bm12B6 groups in these experiments showed even higher levels of
anti-chromatin, RF, anti-ssDNA, and anti-dsDNA at later
time points (Fig. 2B). The pattern of a gradual falloff in
titer over time was unchanged if the analysis included data only from
mice that survived the entire experiment (not shown). In addition,
anti-cathepsin C and anti-collagen IV autoantibodies also
showed the same pattern as anti-ssDNA in both the CD4KO and CD8KO
experiments (data not shown). Striking increases in total serum IgM and
IgG were seen in the cGVH groups (bm12B6 and bm12CD8KO). The
bm12CD4KO group showed a modest, transient (but significant)
elevation of total IgM and IgG at weeks 2, 4, and 8 (Fig. 3).
|
|
Proteinuria was measured at 2- to 4-wk intervals in female
recipients to assess renal involvement during cGVH disease. As shown in
Fig. 4, bm12CD4KO and B6CD4KO mice
failed to show any significant elevation of renal protein excretion,
while concomitant bm12B6 mice showed substantial levels. In
contrast, bm12CD8KO mice showed elevated proteinuria levels
comparable to the concomitant positive control (bm12B6 group).
|
. The mean values of
all kidney scores of bm12B6 mice in the CD4KO experiments were
significantly higher than those of bm12CD4KO or those of B6CD4KO
mice (p < 0.005), while the latter two groups
did not differ (p > 0.05). The light
microscopy scores of the bm12B6 and bm12CD8KO were comparably
abnormal, but surprisingly, so were those of B6CD8KO mice. However,
B6CD8KO mice showed normal kidney immunofluorescence scores for IgG
and C3 deposition, while bm12CD8KO and bm12B6 mice showed
comparably elevated scores. The histopathologic character of the
glomerular involvement of the B6CD8KO mice was similar to that of
the bm12CD8KO group. Neither unmanipulated CD8KO mice nor control
CD8KOCD8KO mice showed significant renal abnormalities (data not
shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(DBA/2 x
C57BL/6)F1, which has the potential complication of Class I
reactivities. In addition, adult-thymectomized, irradiated, bone
marrow-reconstituted mice do not have T cell deficits that are entirely
comparable to those of the knockout animals in the present study or the
congenitally athymic mice in our previous unpublished work. Thus, the cellular interactions that induce the production of autoantibodies may be much more complicated that we had thought. What role could the endogenous T cells play in this syndrome? They could supply T cell factors in a way that does not require any particular specificity of interaction between T cells and B cells. Alternatively, they could be essential for development of the B cell repertoire that eventually becomes autoreactive. This may entail the selection of particular autoantibody-specific B cells, or it could involve a more general influence on B cell ontogeny that might influence the ability of B cells to respond to allogeneic help. Another possibility is that the endogenous T cells are themselves induced to become autoreactive as a result of interaction with autoreactive B cells that have in turn been activated by interaction with the donor alloreactive T cells. Such a mechanism would parallel the stepwise T-B interactions proposed by Lin, Mamula, and Janeway, in which immunization with foreign and self cytochrome c induces foreign reactive T cells that can provide help for B cells that see both the self and the foreign protein and which, in turn, then can induce the activation of T cells that are reactive with the self protein (20). Such sequential breaking of T and B cell tolerance may be generally applicable to models of systemic autoimmunity in which a spectrum of autoantibodies is made that is largely reactive against nuclear components (21). These possibilities are currently being tested in our laboratory by cell-transfer experiments with purified T cells and B cells.
A recent publication from Merino’s laboratory may help to elucidate our observation (12). These authors found that the ability of B cells to respond in vitro to allogeneic help by proliferation and production of IgG was dependent on the development of such B cells in an environment that had CD4+ T cells. B cells derived from athymic (nu/nu) mice or from mice treated with anti-CD4 mAb failed to be stimulated by MHC-disparate T cells, although they in turn could stimulate such T cells to produce normal amounts of IL-2, IL-4, and IL-10. The B cell defect was reversed by preincubation with IL-4. Whether IL-4 pretreatment or syngeneic B cell reconstitution of CD4KO mice can restore their autoimmune responsiveness to alloreactive T cells in our cGVH model is currently under investigation in our laboratory.
It is formally possible that the essential role of the host CD4 T cells
in the autoimmune cGVH is to react against the Ia+ donor
cells in a host-vs-graft (HVG) reaction. Our previous published work
with this model makes this highly unlikely. We showed that all of the
autoantibodies in the cGVH were made by the host B cells and that we
could detect only very transient evidence for the presence of the donor
B cells in the recipient (4). Therefore, the requirement for CD4 T
cells in the recipient cannot be based on a direct role of donor B
cells in autoantibody formation in this model. In addition, the
experiments in which we compared the parentparent with the
parentF1 protocols for cGVH, utilizing the coisogenic B6
and bm12 strains, showed little difference between these two models
(9). Since we would not expect any HVG recognition in the
parentF1 transfer, it is unlikely that HVG plays an
important role in the parentparent transfer.
We are currently investigating whether the B cells in CD4KO mice have functional abnormalities that may prevent them from responding to allogeneic help by the production of autoantibodies. Various molecules have been recently described as mediators of costimulatory signals necessary for the establishment of an efficient T-B cell collaboration. For instance, after the recognition of specific peptides by T cells, the interaction between CD28 and CTLA-4 with their ligands on B cells, B7-1 and B7-2, is essential to insure T cell activation instead of anergy or apoptosis (22, 23). Signals mediated through CD40 on B cells, which interacts with ligand for CD40 on activated T cells, seem to be required for the generation of germinal centers, Ig-class switch, and rescue from apoptosis of B cells expressing high affinity sIg during the process of somatic mutation (24, 25, 26). Moreover, the interaction of some adhesion molecules, such as LFA-1, with their respective ligands can provide essential accessory signals in many immune reactions (27). In previous work, B cells from athymic CBF1nu/nu mice expressed the above-mentioned molecules, as well as MHC class II, in a manner identical to that of euthymic CB6F1 mice (12, 28). On the other hand, it has also been reported that MHC class II-restricted Ag presentation by Langerhans cells is deficient in athymic nude mice, and that this defect can be restored after a thymus graft (29). It is therefore possible that Langerhans cells and B cells in CD4KO mice are deficient in autoantigen presentation to alloreactive T cells in our model.
The finding that the CD8KO recipients of B6 spleen cells had
significant development of renal disease was unexpected. In three
separate experiments, this group showed histologic glomerular lesions
comparable to those seen in the bm12B6 and bm12CD8KO cGVH groups.
On the other hand, B6CD8KO mice never showed increased levels of
autoantibodies (Fig. 2
), and little Ig deposition was seen in animals
sacrificed 24 wk after spleen cell transfer (Fig. 5). In another
experiment, involving sacrifice at 32 wk, some Ig and complement
deposition was found, although not as much as in the positive cGVH
controls (data not shown). We speculate that the glomerular disease in
these animals represents a failure of normal immunoregulation that
occurs as a result of the transfer of CD8 cells into the CD8KO
recipients. It is also possible that persistent genetic contributions
from the 129 genome in which the CD8KO mutation was first inserted
might code for minor Ags that would be recognized by the donor B6 T
cells in a cGVH reaction that could directly produce glomerular damage.
Even though the B6CD8KO line is backcrossed nine generations to B6, 129
genes closely linked to CD8 would by definition cosesegrate with the
CD8KO locus. In any case, the B6CD8KO group presents a potentially
interesting model of pauciimmune glomerulonephritis which deserves
further study.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Robert A. Eisenberg, Chief, Division of Rheumatology, University of Pennsylvania Medical Center, 502 Maloney, 3600 Spruce Street, Philadelphia, PA 19104-4283. E-mail address:
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; cGVH, chronic graft-vs-host; CD4KO, CD4 knockout mice; CD8KO, CD8 knockout mice; bm12, B6.C-H2bm12/KhEg mice; B6, C57BL/6 mice; RF, rheumatoid factor; BBS, borate-buffered saline; EDF, equivalent dilution factor; HVG, host-vs-graft.
Received for publication April 27, 1998. Accepted for publication July 24, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. Kim, K. Park, H. J. Kim, J. Kim, H.-A Kim, D. Jung, H. J. Kim, H.-J. Choi, S.-Y. Choi, K. W. Seo, et al. Breaking of CD8+ T Cell Tolerance through In Vivo Ligation of CD40 Results in Inhibition of Chronic Graft-versus-Host Disease and Complete Donor Cell Engraftment J. Immunol., November 15, 2008; 181(10): 7380 - 7389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-H. Shao, R. A. Eisenberg, and P. L. Cohen The Mer Receptor Tyrosine Kinase Is Required for the Loss of B Cell Tolerance in the Chronic Graft-versus-Host Disease Model of Systemic Lupus Erythematosus J. Immunol., June 1, 2008; 180(11): 7728 - 7735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhao, L. C. Burkly, S. Campbell, N. Schwartz, A. Molano, A. Choudhury, R. A. Eisenberg, J. S. Michaelson, and C. Putterman TWEAK/Fn14 Interactions Are Instrumental in the Pathogenesis of Nephritis in the Chronic Graft-versus-Host Model of Systemic Lupus erythematosus J. Immunol., December 1, 2007; 179(11): 7949 - 7958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Herber, T. P. Brown, S. Liang, D. A. Young, M. Collins, and K. Dunussi-Joannopoulos IL-21 Has a Pathogenic Role in a Lupus-Prone Mouse Model and Its Blockade with IL-21R.Fc Reduces Disease Progression J. Immunol., March 15, 2007; 178(6): 3822 - 3830. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Ma, F. Chen, M. P. Madaio, P. L. Cohen, and R. A. Eisenberg Modulation of Autoimmunity by TLR9 in the Chronic Graft-vs-Host Model of Systemic Lupus Erythematosus J. Immunol., November 15, 2006; 177(10): 7444 - 7450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Witsch, H. Cao, H. Fukuyama, and M. Weigert Light chain editing generates polyreactive antibodies in chronic graft-versus-host reaction J. Exp. Med., July 10, 2006; 203(7): 1761 - 1772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zhang, I. Todorov, Z. Zhang, Y. Liu, F. Kandeel, S. Forman, S. Strober, and D. Zeng Donor CD4+ T and B cells in transplants induce chronic graft-versus-host disease with autoimmune manifestations Blood, April 1, 2006; 107(7): 2993 - 3001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen, D. Perry, S. A. Boackle, E. S. Sobel, H. Molina, B. P. Croker, and L. Morel Several Genes Contribute to the Production of Autoreactive B and T Cells in the Murine Lupus Susceptibility Locus Sle1c J. Immunol., July 15, 2005; 175(2): 1080 - 1089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Choudhury, M. A. Maldonado, P. L. Cohen, and R. A. Eisenberg The Role of Host CD4 T Cells in the Pathogenesis of the Chronic Graft-versus-Host Model of Systemic Lupus Erythematosus J. Immunol., June 15, 2005; 174(12): 7600 - 7609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kim, W. S. Choi, S. La, J.-H. Suh, B.-S. Kim, H. R. Cho, B. S. Kwon, and B. Kwon Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activation-induced cell death of donor CD4+ T cells Blood, March 1, 2005; 105(5): 2206 - 2213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Sekiguchi, R. A. Eisenberg, and M. Weigert Secondary Heavy Chain Rearrangement: A Mechanism for Generating Anti-double-stranded DNA B Cells J. Exp. Med., January 6, 2003; 197(1): 27 - 39. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kanamaru, A. Nakao, I. Shirato, K. Okumura, H. Ogawa, Y. Tomino, and C. Ra Chronic Graft-versus-Host Autoimmune Disease in Fc Receptor {gamma} chain-deficient Mice Results in Lipoprotein Glomerulopathy J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1527 - 1533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Sekiguchi, S. M. Jainandunsing, M. L. Fields, M. A. Maldonado, M. P. Madaio, J. Erikson, M. Weigert, and R. A. Eisenberg Chronic Graft-Versus-Host in Ig Knockin Transgenic Mice Abrogates B Cell Tolerance in Anti-Double-Stranded DNA B Cells J. Immunol., April 15, 2002; 168(8): 4142 - 4153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Maloney, B. M. Sandmaier, S. Mackinnon, and J. A. Shizuru Non-Myeloablative Transplantation Hematology, January 1, 2002; 2002(1): 392 - 421. [Abstract] [Full Text]
|
|
|
|
|
|
N. Feuerstein, F. Chen, M. Madaio, M. Maldonado, and R. A. Eisenberg Induction of Autoimmunity in a Transgenic Model of B Cell Receptor Peripheral Tolerance: Changes in Coreceptors and B Cell Receptor-Induced Tyrosine-Phosphoproteins J. Immunol., November 15, 1999; 163(10): 5287 - 5297. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
