The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yasuoka, H.
Right arrow Articles by Kuwana, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yasuoka, H.
Right arrow Articles by Kuwana, M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Nucleotide
*OMIM*Protein
*UniGene
Medline Plus Health Information
*Scleroderma
The Journal of Immunology, 2003, 171: 6883-6890.
Copyright © 2003 by The American Association of Immunologists

A Novel Protein Highly Expressed in Testis Is Overexpressed in Systemic Sclerosis Fibroblasts and Targeted by Autoantibodies 1

Hidekata Yasuoka*,{dagger}, Hironobu Ihn{ddagger}, Thomas A. Medsger, Jr§, Michito Hirakata*, Yutaka Kawakami{dagger}, Yasuo Ikeda* and Masataka Kuwana2,{dagger}

* Department of Internal Medicine and {dagger} Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; {ddagger} Department of Dermatology, University of Tokyo Graduate School of Medicine, Tokyo, Japan; and § Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nearly all autoantibody specificities in sera from patients with systemic sclerosis (SSc) target proteins distributed ubiquitously, and Abs against proteins whose expression is restricted to the affected sites have not been identified. In this study we describe SSc-specific autoantibody to a novel testicular Ag, termed protein highly expressed in testis (PHET), which is ectopically overexpressed in SSc dermal fibroblasts. A partial cDNA encoding PHET was isolated by immunoscreening of a HepG2 cDNA library with an SSc serum. PHET appeared to be a member of the UniGene cluster Hs.129872, but had a unique exon composition and a characteristic mRNA expression profile restricted to the testis. Serum Abs to a recombinant PHET fragment were detected in nine (8.4%) of 107 SSc patients, but in none of 50 systemic lupus erythematosus patients or 77 healthy controls. In SSc patients, the presence of anti-PHET Abs was associated with diffuse cutaneous SSc and lung involvement (p = 0.02 and 0.01, respectively). PCR-based quantitative analysis of PHET mRNA expression in cultured dermal fibroblasts showed increased expression of PHET mRNA in SSc fibroblasts compared with control fibroblasts. PHET-reactive Abs purified from SSc sera stained the cytoplasm of SSc dermal fibroblasts, and the staining intensity tended to be more prominent on SSc compared with control fibroblasts. These findings suggest that the autoantibody response to PHET can be induced by ectopic overexpression of PHET in dermal fibroblasts in SSc patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) 3 is a disease characterized by excessive fibrosis of the skin and internal organs as well as microvascular injury (1). One of the immunologic features in SSc patients is the presence of serum autoantibodies to various nuclear Ags, including Scl-70/topoisomerase I (topo I), centromere/kinetochore, and RNA polymerase (RNAP) I and III (1, 2, 3). These SSc-related anti-nuclear Abs target autoantigens expressed by all tissues and cell types. Although these Abs are highly specific to SSc and are associated with distinct SSc clinical subsets (1, 2, 3), the mechanism for their production remains unclear.

Excessive fibrosis of the skin and internal organs in SSc patients is shown to be mediated by fibroblasts that acquire the capacity of enhanced cellular proliferation and production of an excessive amount of extracellular matrix (4, 5). However, phenotypic and functional changes in affected fibroblasts and autoantibody responses in SSc patients have been studied separately, and there is little evidence that links these two characteristic phenomena. A recent microarray analysis of SSc dermal fibroblasts found that SSc-related autoantigens, including centromeric protein B, fibrillarin, a largest subunit of RNAP II, and topo I, were up-regulated in SSc dermal fibroblasts obtained from affected skin compared with those from unaffected skin, suggesting a possible association between overexpression of SSc-specific autoantigens in the affected fibroblasts and the specific autoantibody responses (6). In contrast, autoantibodies reactive with dermal fibroblasts have been reported in sera from SSc patients (7, 8, 9, 10), but fibroblast-specific autoantigens have not been identified to date.

One of our recent projects was to isolate cDNAs encoding subunits of RNAP I/III (11). For this purpose, a cDNA library constructed from hepatoma cell line HepG2 was screened using an SSc serum positive for a high titer of anti-RNAP I/III Abs. One of isolated clones had a characteristic mRNA expression profile restricted to the testis, and a protein encoded by this novel cDNA fragment was termed protein highly expressed in testis (PHET). By homology search in the genetic database, PHET cDNA was found to be a member of the UniGene cluster Hs.129872 encoded by chromosome 17q21, but had a unique exon composition. In this study we have evaluated clinical correlates of serum anti-PHET autoantibodies and possible underlying mechanisms for this autoantibody response by examining mRNA and protein expression of PHET in SSc dermal fibroblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and controls

We examined 107 Japanese patients with SSc who were followed at Keio University Hospital. All patients fulfilled the American College of Rheumatology (formerly American Rheumatism Association) preliminary classification criteria for SSc (12). Eighty-nine percent were female, the mean age at disease onset was 44.0 ± 13.8 years, and 31% were classified as having diffuse cutaneous SSc. None of the male patients had received a vasectomy. Sera from 50 patients with systemic lupus erythematosus (SLE) who met the American College of Rheumatology revised criteria (13) and those from 77 healthy individuals were studied as controls.

For cDNA library screening, we used serum from SSc patient FA, who was followed at University of Pittsburgh. FA was a 73-year-old Caucasian female who had diffuse cutaneous SSc with rapidly progressive skin thickening and was positive for serum anti-RNAP I/III Abs. She had severe myocardial involvement and eventually died of heart failure.

Skin biopsies from a clinically affected skin area (dorsal forearm) were available in nine patients with early diffuse cutaneous SSc (two men and seven women). As controls, skin biopsies were obtained from a clinically and pathologically unaffected area of 13 patients with nonconnective tissue diseases.

All serum and skin biopsy specimens were collected after the patients and controls gave their written informed consent approved by individual institutional review boards.

Clinical and serologic features

Clinical and laboratory findings were systematically evaluated and recorded for all 107 SSc patients. The disease onset was defined as the time of the first symptom attributable to SSc. SSc patients were classified as having diffuse cutaneous SSc, limited cutaneous SSc, or SSc in overlap, according to published guidelines (1, 14). The definition used to describe organ involvement (joint, vasculature, esophagus, heart, kidney, lung, and pulmonary artery) was described previously (2). In this system, lung involvement indicates pulmonary interstitial fibrosis on chest radiograph, whereas pulmonary artery involvement indicates clinical evidence of isolated pulmonary arterial hypertension and increased mean pulmonary arterial pressure documented by echocardiogram or right heart catheterization. To identify eight SSc-related autoantibodies, including Abs to topo I, centromere, RNAP I/III, U1 ribonucleoprotein (RNP), U3 RNP, Th/To, PM-Scl, and Ku (2), all SSc sera were analyzed by indirect immunofluorescence using commercially prepared slides of monolayer HEp-2 cells (MBL, Nagano, Japan) as well as RNA and protein immunoprecipitation assays (15).

Human dermal fibroblast cultures

Primary cultures of dermal fibroblasts were established from skin biopsies (16). Each fibroblast culture was maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 50 mg/ml amphotericin B as monolayer cells in a humidified atmosphere of 5% CO2 at 37°C and were analyzed between the third and sixth subpassages. The culture period from the time of biopsy was principally matched between SSc and control fibroblasts to avoid the influence of senescence. Fibroblasts were grown to semiconfluence and then incubated for 24 h in serum-free medium (QBSF51; Sigma-Aldrich, St. Louis, MO) before use in mRNA and protein expression analysis.

Complementary DNA library screening

Plaques (5 x 105) of a randomly primed HepG2 {lambda}gt11 cDNA library (Clontech Laboratories, Palo Alto, CA) were screened using SSc serum FA as described previously (17). Positive clones were isolated, and their nucleotide sequences were determined on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster, CA) using the BigDye Terminator Cycle Sequences Ready Reaction kit (Applied Biosystems) according to the manufacturer’s protocol. The nucleotide sequence of the isolated cDNA was analyzed using genetic databases at the National Center for Biotechnology Information (NCBI).

Detection and quantification of PHET mRNA expression

mRNA expression for PHET, Hs.129872 variants, and {beta}-actin in various human tissues and cultured dermal fibroblasts was evaluated using RT-PCR. Total RNAs obtained from a panel of normal human tissues were purchased from Clontech Laboratories, and total RNAs from cultured dermal fibroblasts and HepG2 cells were isolated using the RNeasy kit (Qiagen, Valencia, CA). A first-strand cDNA was synthesized from each total RNA sample with an oligo(dT)12–15 primer using AMV reverse transcriptase XL (Takara, Kusatsu, Japan). mRNA for PHET and the variants were detected by PCR using cDNA (12.5 ng total RNA equivalent) as a template. A primer set (forward, 5'-GGATTGCTTACACCTGATGC-3'; reverse, 5'-TTTGGTGGGAGTGCTTGAAC-3') was designed to amplify both PHET and the Hs.129872 variants lacking exon 8 in different sizes (465 and 423 bp, respectively). The PCR conditioning was 3 min at 94°C, followed by 30 or 35 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. A control PCR for {beta}-actin was conducted for 25 cycles using a human {beta}-actin control amplimer set (Clontech Laboratories). PCR products were fractionated on 2% agarose gels and stained with ethidium bromide. In some experiments the intensity of individual DNA bands corresponding to PHET, exon 8- variants, and {beta}-actin on ethidium bromide-stained gels was semiquantified by densitometry using Molecular Imager FX (Bio-Rad, Hercules, CA). Relative expression levels for PHET and exon 8- variants were normalized based on the corresponding {beta}-actin band intensity.

mRNA expression for PHET and {beta}-actin in cultured fibroblasts was further quantified using TaqMan PCR (Applied Biosystems). This system is a comparative RT-PCR following the theory that the quantity of target mRNA is proportional to its PCR products during the logarithmic amplification phase (18). A combination of primers and a probe specific for PHET was designed using Primer-Express software (Applied Biosystems) as follows: forward primer, 5'-CCAGCCTGAAGGTCAGCAATAG-3'; reverse primer, 5'-CGCCTTGGCTAACATCAGAA-3'; and probe, 5'-(FAM)-TCAGAAGGCTGTAGAACAGGAGGATGAGC-(TAMRA)-3'. A forward primer and a probe contained the exon 8 sequence present in PHET, but not in the variants. Each cDNA sample (25 ng of total RNA equivalent) were amplified in duplicate in the presence of 300 nM of each primer and probe, 0.5 U of AmpErase uracil N-glycosylase, and 1.25 U AmpliTaq Gold (Applied Biosystems). The PCR conditioning was 2 min at 50°C and 10 min at 95°C, followed by 60 cycles of 15 s at 95°C and 1 min at 60°C. {beta}-Actin mRNA expression was also quantitatively assessed as a control using TaqMan {beta}-actin control reagent (Applied Biosystems). Relative expression levels for PHET mRNA were calculated based on the standard curve obtained by serial dilutions of testis cDNA and adjusted by the level of {beta}-actin expression.

Immunoblots

Anti-PHET Abs were detected by immunoblots using recombinant PHET (rPHET) that was expressed as a maltose-binding protein (MalBP) fusion protein as previously described (19). The rPHET encoded 203 aa residues corresponding to the fragment isolated by cDNA library screening. rPHET/exon 8- (rPHET/Ex8-), a recombinant polypeptide that corresponded to the portion encoded by rPHET, but lacked 14 aa residues encoded by exon 8, was also expressed as a MalBP fusion protein. Briefly, DNA fragments amplified from testis cDNA were subcloned in-frame into the pMAL-c2 expression vector (New England Biolabs, Beverly, MA) and were transformed into a competent Escherichia coli strain DH5{alpha} (Toyobo, Osaka, Japan). Nucleotide sequences of both strands of each DNA construct were determined to verify the translational frames and insert sequences. The expression of recombinant MalBP fusion protein was induced by isopropyl-{beta}-D-thiogalactopyranoside. A bacterial lysate containing rPHET or rPHET/Ex8- was fractionated on 7.5% polyacrylamide-SDS gels and transferred onto nitrocellulose membranes. A portion of the membranes was stained with Amido Black, and the remaining membranes were blocked with 5% nonfat milk and incubated with rabbit anti-MalBP polyclonal Abs (New England Biolabs) diluted at 1/10,000 or serum samples diluted at 1/250 or 1/1,000 that were preincubated with a bacterial lysate expressing MalBP to remove Abs reactive with bacterial components and MalBP. After incubation with goat anti-human or anti-rabbit IgG Abs conjugated to alkaline phosphatase (Cappel, Aurora, OH), the membranes were visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

To evaluate the expression of PHET protein in cultured fibroblasts, total cellular lysates (1.6 x 104 cells/lane) were fractionated on 6% polyacrylamide-SDS gels, transferred onto nitrocellulose membranes, and incubated with anti-PHET-positive SSc sera (1/100) or rabbit anti-actin polyclonal Abs (1/200; Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with goat anti-human or rabbit IgG Abs conjugated with alkaline phosphatase (Cappel), the membranes were visualized with a substrate.

In some experiments anti-PHET-positive sera were pretreated with a bacterial lysate containing rPHET or rPHET/Ex8- before applying them to immunoblots.

Indirect immunofluorescence on cultured dermal fibroblasts

Protein expression of PHET in cultured dermal fibroblasts was examined by indirect immunofluorescence using anti-PHET-positive SSc sera as a probe. Briefly, SSc or control dermal fibroblasts (1 x 103) were spread on fibronectin-coated, eight-well culture slides (BIOCOAT; BD Biosciences, Bedford, MA) and cultured for 24 h in complete medium, followed by treatment with serum-free medium for 12 h. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature or a 50% ethanol-50% acetone mixture for 5 min at 4°C. Fixed cells were then incubated with serum samples diluted at 1/20 or rPHET-reactive Abs that were purified from anti-PHET-positive SSc sera by binding to rPHET on preparative immunoblots, followed by elution at acid pH (20). After incubation with FITC-conjugated goat anti-human IgG Abs (MBL, Nagano, Japan) for 30 min, the slides were mounted and examined with a confocal laser fluorescent microscope (LSM5 PASCAL; Carl Zeiss, Göttingen, Germany).

Statistical analyses

Difference in frequencies was analyzed using the {chi}2 test or Fisher’s two-tailed exact test when applicable. The mean values were compared between two groups using Mann-Whitney U test. The correlation coefficient was determined using a single regression model.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of PHET cDNA

By immunoscreening of a HepG2 cDNA library using an SSc serum FA, 23 cDNA clones were isolated. One of these clones (9p) was further characterized because of several interesting features as described below. A complete nucleotide sequence of 9p and its deduced amino acid sequence of the predicted open reading frame are shown in Fig. 1A. Clone 9p encoded 204 aa without a stop codon, and it was apparently partial. A homology search on the NCBI database revealed that nucleotide sequence of 9p was highly homologous to those of cDNA fragments grouped in the UniGene cluster Hs.129872; SPAG9 Sperm-associated Ag 9 (Fig. 1B). This gene cluster is encoded by chromosome 17q21, and nine reported cDNA fragments are thought to be originated from the same gene as alternative splicing products (updated on May 10, 2003). When the nucleotide sequence of our clone was compared with the reported sequences, 9p had an additional 42-bp sequence (Fig. 1A). This insertion sequence was likely to be an exon (exon 8), because an identical sequence was found in chromosome 17q21 in a correct order as well as in an another cDNA fragment (sperm-associated Ag 9 clone MGC:14967) in the Hs.129872. In addition, a typical splicing motif sequence was found upstream of the insertion sequence in the genome. As a result, 9p had a unique exon composition consisting of exons 7–8-10–11-12. Sperm-associated Ag 9 clone MGC:14967 also had exon 8, but exon 8 was connected to exon 9, which was specifically used by this variant. Exon 8 was not used by other reported cDNA fragments in the Hs.129872. To obtain longer cDNA covering the 9p sequence, testis cDNA was amplified using a series of primer sets corresponding to exons used by other variants. The longest cDNA isolated at the moment is 2369-bp fragment consisting of exons 5, 7, 8, and 10–26. This cDNA has a sequence completely matched to mRNA for sperm protein (HSS) (21), but has an additional exon 8.



View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 1. A, A complete nucleotide sequence and its deduced amino acid sequence of clone 9p, a partial cDNA fragment of PHET. Numbers on the left denote bases from the 5' terminus and amino acid residues from the N terminus. A unique nucleotide sequence encoded by exon 8 is underlined. The locations of forward and reverse primers used to detect PHET mRNA are shown as arrows. B, Predicted exon composition of PHET (9p and a longer fragment) and the reported sequences in the UniGene cluster Hs.129872. Each box on the chromosome 17q21 shows an exon predicted based on comparison between genomic sequence and the cDNA sequences. A closed box in cDNA FLJ34602 fis is a portion not encoded by chromosome 17. • and {circ}, Initiation and stop codons, respectively, of predicted open-reading frames of individual cDNA sequences. Arrows denote locations of forward and reverse primers used to evaluate mRNA expression. The RT-PCR product size in individual cDNAs was predicted based on their exon composition. NA, no amplification.

 
When the mRNA expression profile of Hs.129872 was surveyed in the NCBI database, various tissues and cell lines were reported as cDNA sources for this gene cluster. Serial analysis of gene expression tags corresponding to the Hs.129872 were again isolated from libraries constructed from various tissues and cell lines. These findings indicate that transcripts of the Hs.129872 are expressed ubiquitously when all splice variants are combined. To examine the mRNA expression profile of 9p, RT-PCR (30 cycles) was conducted using a panel of cDNAs derived from normal human tissues. To discriminate PCR products of 9p and other splice variants in the Hs.129872, we designed a forward primer in exon 7 and a reverse primer in a junction of exons 11 and 12 (Fig. 1A). The predicted PCR product sizes obtained from individual cDNA fragments are listed in Fig. 1B. As shown in Fig. 2, a 465-bp product corresponding to 9p (exons 7–8-10–11-12) was amplified exclusively in testis among normal tissues, whereas a 423-bp product corresponding to exons 7–10-11–12 was amplified in placenta, testis, small intestine, brain, spleen, and liver. The HepG2 cell line, an original mRNA source of 9p, produced both bands, but a 423-bp band was weak. When the number of cycles in PCR was increased to 35, a faint band for 9p was detected in stomach, placenta, and brain in addition to testis, whereas a 423-bp band was detected ubiquitously (data not shown). Based on a unique exon composition and a restricted mRNA expression profile, a protein encoded by clone 9p was termed PHET.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 2. The mRNA expression of PHET and Hs.129872 variants lacking exon 8 in various normal tissues as well as in the HepG2 cell line. PCR was performed for 30 cycles using a primer set designed to amplify both PHET and exon 8- variants, which can be discriminated based on the sizes of the products (465 and 423 bp, respectively). PCR products were fractionated on 2% agarose gels and stained with ethidium bromide. {beta}-Actin mRNA was also amplified as a control (838 bp).

 
Detection of serum anti-PHET Abs in SSc sera

A partial fragment of PHET (204 aa residues) was expressed as a recombinant MalBP-fusion protein (rPHET) and used in immunoblotting. In SDS-PAGE, rPHET yielded a molecular mass of ~90 kDa, which was larger than the size predicted from the amino acid number. This is probably due to the highly acidic nature of this polypeptide, leading to a slow migration on gels. As shown in Fig. 3A, serum FA, used as a probe in the cDNA library screening, as well as anti-MalBP polyclonal Abs strongly reacted with intact rPHET and its degradation products. When Abs to PHET were screened in sera (1/250) from 107 Japanese patients with SSc by immunoblots, nine (8.4%) sera showed reactivity to rPHET. All sera positive for anti-PHET Abs bound rPHET when sera were diluted at 1/1000. In contrast, none of 50 SLE sera or 77 healthy control sera (1/250) reacted with rPHET. The frequency of serum anti-PHET Ab was significantly higher in SSc patients than in SLE patients and healthy controls (p = 0.03 and 0.007, respectively).



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3. A, Ab reactivities to rPHET and rPHET/Ex8- detected by immunoblots in representative SSc sera. A bacterial lysate containing rPHET or rPHET/Ex8- was fractionated on 7.5% polyacrylamide-SDS gels, transferred onto nitrocellulose membranes, and then probed with serum FA used as a probe in the cDNA library screening (lane 1), SSc sera (lanes 2–10), and anti-MalBP polyclonal Abs (lane 11). SSc sera in lanes 2–6 were positive for anti-PHET Abs, whereas SSc sera in lanes 7–10 were negative. Molecular mass markers (Mr) and a bacterial lysate containing recombinant proteins (Ly) were stained with Amido Black. B, Detection of Abs reactive with the PHET-specific sequence in representative SSc sera positive for anti-PHET Abs. Three SSc sera that reacted with rPHET (lanes 1–3) were preincubated with a bacterial lysate containing rPHET/Ex8- and applied to immunoblots using rPHET (rP) and rPHET/Ex8- (rV) as Ags. Molecular mass markers (Mr) and a bacterial lysate containing recombinant proteins were stained with Amido Black.

 
Ab reactivity to rPHET/Ex8- was examined by immunoblots in the nine SSc sera positive for anti-PHET Abs (Fig. 3A). All but one anti-PHET-positive serum reacted with rPHET/Ex8-. Serum HY reacted with rPHET, but not with rPHET/Ex8-, indicating that a determinant containing the PHET-specific exon 8 sequence was recognized by this serum. Recombinant PHET/Ex8- was not recognized by four randomly selected SSc sera lacking reactivity to rPHET. To further evaluate the presence of Abs reactive with the PHET-specific sequence, eight sera reactive with both rPHET and rPHET/Ex8- were preincubated with a bacterial lysate containing rPHET/Ex8- and applied to immunoblots (Fig. 3B). SSc serum OdY recognized rPHET after the absorption of Abs reactive with rPHET/Ex8-, but the remaining seven sera lost their reactivity to rPHET. Based on these findings, among nine SSc sera with anti-PHET Abs, seven sera solely had Abs reactive with determinants shared by both rPHET and rPHET/Ex8-, one serum (HY) exclusively had Abs specific to the PHET-specific exon 8 sequence, and the remaining serum (OdY) had both specificities. It was interesting to note that both SSc patients with Abs against the PHET-specific sequence were males, whereas the remaining seven patients were all females. The prototype serum obtained from female patient FA also had Abs reactive with the shared sequence alone.

Clinical characteristics associated with anti-PHET Abs

Demographic and clinical findings as well as coexistent SSc-related Abs were compared between the nine SSc patients with anti-PHET Ab and the 98 SSc patients without anti-PHET Ab (Table I). Six of nine anti-PHET-positive patients had diffuse cutaneous SSc, and this frequency was significantly higher than that in anti-PHET-negative patients (p = 0.02). The frequencies of all SSc-related organ involvements except pulmonary artery involvement (isolated pulmonary arterial hypertension) tended to be increased in anti-PHET-positive compared with anti-PHET-negative SSc patients, and lung involvement (pulmonary interstitial fibrosis) was significantly more frequently found in SSc patients with anti-PHET Abs than those without anti-PHET Abs (p = 0.01). In this regard, the prototype patient FA had diffuse cutaneous SSc accompanied by severe lung and heart involvement. These findings suggest a relationship between the anti-PHET Ab response and more extensive fibrotic changes in SSc patients. Eight of nine patients positive for anti-PHET Abs had additional SSc-related Abs, including anti-topo I, anticentromere, and anti-RNAP I/III Abs, but there was no significant difference in the frequencies of SSc-related Abs between SSc patients with and without anti-PHET Abs.


View this table:
[in this window]
[in a new window]
  		Table I. Demographic and clinical findings and SSc-related autoantibody profiles in SSc patients with or without serum anti-PHET Abs

 
PHET mRNA expression in SSc dermal fibroblasts

The autoantibody response to PHET was detected in both male and female patients with SSc, although PHET mRNA was almost exclusively expressed in the testis among normal tissues. As PHET mRNA was expressed in the majority of cancer and transformed cell lines (our unpublished observations), we hypothesized that PHET is expressed in SSc dermal fibroblasts that have some transformed properties. To test this hypothesis, the expression of mRNAs for PHET and Hs.129872 variants lacking exon 8 was examined in cultured dermal fibroblasts derived from nine SSc patients including two with anti-PHET Abs and control dermal fibroblasts from 13 non-SSc individuals. As shown in Fig. 4A, mRNAs for PHET and exon 8- variants were expressed by all cultured fibroblasts regardless of the presence or the absence of SSc. When mRNA expression levels of PHET and exon 8- variants were compared between SSc and control fibroblasts using semiquantitative analysis by densitometry, relative mRNA expression levels of PHET were significantly higher in SSc fibroblasts than in control fibroblasts (p = 0.0007; Fig. 4B). A patient representing the highest PHET mRNA expression was positive for serum anti-PHET Abs. The expression level of mRNA for exon 8- variants was also significantly higher in SSc fibroblasts compared with control fibroblasts (p = 0.02).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 4. Messenger RNA expression for PHET and Hs.129872 variants lacking exon 8 (exon 8- variants) in cultured SSc and control dermal fibroblasts. A, cDNAs isolated from nine SSc and 13 control fibroblast lines as well as cDNA from testis and HepG2 were subjected to PCR to amplify both PHET and exon 8- variants. The PCR products were fractionated on 2% agarose gels and stained with ethidium bromide. The sizes for PHET, exon 8- variants, and {beta}-actin products are 465, 423, and 838 bp, respectively. B, Relative mRNA expression levels for PHET and exon 8- variants in nine SSc and 13 dermal fibroblasts were semiquantitatively analyzed using RT-PCR, followed by densitometry. The levels of mRNA expression were normalized based on the {beta}-actin expression level. In SSc samples: {blacktriangleup}, fibroblasts derived from anti-PHET-positive patients; {triangleup}, those from anti-PHET-negative patients. Comparisons were made by nonparametric Mann-Whitney U test.

 
Expression levels of PHET and {beta}-actin mRNAs were further assessed using TaqMan quantitative PCR in seven SSc fibroblasts and nine control fibroblasts. Representative real-time plots of the amounts of amplified products in SSc and control fibroblasts are shown in Fig. 5A. The amount of the PHET product was consistently greater in SSc fibroblasts compared with control fibroblasts, whereas that of the {beta}-actin product showed no difference. As shown in Fig. 5B, relative mRNA expression level of PHET was significantly higher in SSc fibroblasts than in control fibroblasts (p = 0.02). There was no difference in the PHET mRNA expression level between fibroblasts derived from male and female individuals. The relative mRNA expression levels of PHET determined using two different PCR-based methods were significantly correlated with each other (r2 = 0.73; p = 0.00003)



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 5. PHET mRNA expression levels determined by quantitative TaqMan PCR in cultured SSc and control dermal fibroblasts. A, Real-time plots of the amounts of PCR products corresponding to PHET and {beta}-actin in representative SSc and control fibroblasts. B, The relative PHET mRNA expression level in individual fibroblasts was calculated based on the standard curve generated by the amount of PCR products against the amount of input testis cDNA, and adjusted by the {beta}-actin expression. In SSc samples: {blacktriangleup} and {triangleup}, fibroblasts obtained from anti-PHET-positive and -negative patients, respectively. Comparisons were made by nonparametric Mann-Whitney U test.

 
Expression of PHET protein in cultured dermal fibroblasts

To examine whether anti-PHET Abs in patients’ sera bound to dermal fibroblasts, the prototype serum FA was used to stain SSc fibroblasts with increased PHET mRNA expression by indirect immunofluorescence. As shown in Fig. 6A, serum FA produced diffuse cytoplasmic staining in addition to a strong nuclear staining whose pattern was compatible with that produced by anti-RNAP I/III Abs (15). In contrast, healthy control serum did not stain SSc fibroblasts. Because serum FA had coexistent anti-RNAP I/III Abs, Abs reactive with rPHET were affinity purified from serum FA and used in indirect immunofluorescence. The rPHET-reactive Abs showed profound cytoplasmic staining without apparent nuclear staining, whereas no staining was detected with mock-eluted Abs. Similar findings were obtained from two additional rPHET-reactive Abs isolated from anti-PHET-positive SSc sera, including serum HY, which exclusively contained PHET-specific Abs. The rPHET-reactive Abs eluted from serum FA were used to stain additional SSc and control fibroblasts as a probe. The rPHET-reactive Abs stained the cytoplasm of two additional SSc fibroblasts with increased PHET mRNA expression (Fig. 6A, e and g) and two independent control fibroblasts (Fig. 6A, f and h), but the staining intensity was much stronger on SSc fibroblasts than control fibroblasts. An identical finding was obtained when Abs to the PHET-specific sequence eluted from serum HY were used as a probe.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 6. A, Binding of anti-PHET Abs to cultured dermal fibroblasts using indirect immunofluorescence. Cultured fibroblasts were fixed and incubated with sera diluted at 1/20 or rPHET-reactive Abs purified from patients’ sera. After incubation with FITC-conjugated anti-human IgG Abs, the cells were examined under a confocal laser fluorescent microscope. SSc fibroblasts were stained with serum FA (a), healthy control serum (b), rPHET-reactive Abs eluted from serum FA (c), or mock-eluted Abs (d). Fibroblasts derived from two SSc patients (e and g) and two control individuals (f and h) were stained with rPHET-reactive Abs purified from serum FA (original magnification, x200). B, PHET and Hs.129872 variants expressed by SSc and control cultured dermal fibroblasts. Total cellular lysates from SSc fibroblasts (lanes 1 and 2) and control fibroblasts (lanes 3 and 4) were fractionated on 6% polyacrylamide-SDS gels, transferred onto nitrocellulose membranes, and then probed with anti-PHET-positive SSc serum or anti-actin polyclonal Abs. *, An 85-kDa protein whose reactivity was lost after preincubation of SSc sera with rPHET.

 
When total cellular lysates of SSc and control cultured dermal fibroblasts were used as Ag in immunoblots, anti-PHET-positive SSc sera consistently reacted with several proteins with a molecular mass of 80–95 kDa (Fig. 6B). These proteins were not recognized by anti-PHET-negative SSc sera and were likely to be a group of polypeptides encoded by Hs.129872. Of these, an 85-kDa protein appeared to correspond to the PHET protein, because Ab reactivity was lost after preincubation of anti-PHET-positive SSc sera with rPHET. The expression of an 85-kDa protein appeared to be increased in SSc compared with control dermal fibroblasts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified a novel SSc-specific autoantigen PHET by immunoscreening of a HepG2 cDNA library with an SSc serum. In contrast to known SSc-specific autoantigens expressed ubiquitously, PHET is almost exclusively expressed in the testis among normal tissues. Serum anti-PHET Abs were preferentially detected in a subset of SSc patients with extensive skin and lung involvement, although the positive frequency in SSc patients was low (8.4%). Interestingly, mRNA and protein expression of PHET was up-regulated in SSc compared with control dermal fibroblasts, suggesting a causal relationship between the specific autoantibody response and overexpression of the autoantigens in dermal fibroblasts in SSc patients.

A homology search of PHET cDNA on the NCBI database revealed that PHET is encoded by chromosome 17q21 and should be included in the gene cluster Hs.129872. Although many splice variants of Hs.129872 are reported in various human tissues and cell lines, PHET can be discriminated from previously reported cDNA fragments in the Hs.129872, based on a unique exon composition and a preferential mRNA expression in the testis. Isolation of a full-length PHET cDNA is still underway, but our repeated attempts resulted in isolation of a 2369-bp fragment encompassing 781-aa residues (see Fig. 1B). However, it is possible that there are several distinct splice variants containing a PHET-specific sequence.

Because the majority of anti-PHET-positive sera reacted with rPHET/Ex8-, one can argue that one of the Hs.129872 sequences lacking exon 8 is a real target recognized by SSc sera, and PHET cDNA was isolated due to the cross-reactivity between PHET and other variants. In this regard we found two SSc sera containing Ab to the PHET-specific exon 8 sequence, and one of them exclusively had PHET-specific Ab. Therefore, we believe that an actual target of the autoantibody response in SSc patients is PHET rather than the variants lacking exon 8.

PHET mRNA and protein were expressed by cultured dermal fibroblasts regardless of the presence or the absence of SSc. However, increased expression of PHET mRNA in SSc fibroblasts compared with control fibroblasts was demonstrated by two different methods, including RT-PCR followed by densitometry and the TaqMan quantitative PCR, whereas mRNA expression of the variants lacking exon 8 was also up-regulated in SSc compared with control fibroblasts. By using rPHET-reactive Abs purified from SSc sera as a probe, we demonstrated that protein expression of PHET tended to be increased in SSc compared with control fibroblasts. It is interesting to use a pair of fibroblasts obtained from affected and unaffected areas of the same SSc patient to confirm this finding. However, the expression of PHET mRNA in cultured fibroblasts is inconsistent with its restricted expression profile in the testis in a panel of normal tissues. This might be explained by a small number of residential fibroblasts in normal tissues. Another possibility is secondary changes in the gene expression profile of fibroblasts that were cultured in the presence of various serum factors (22). Further studies investigating in situ expression of PHET in skin biopsies of SSc patients are necessary to confirm our findings. This study is currently ongoing, but generation of the PHET-specific Abs and probes is difficult because of a high homology between PHET and other Hs.129872 transcripts.

It is unclear whether anti-PHET autoantibody responses contribute to the disease process or result from the pathologic process. The basic mechanisms regulating autoantibody responses in SSc patients remain unclear, but our recent studies examining the cellular mechanism controlling anti-topo I Ab production have indicated that production of this autoantibody results from an Ag-driven and T cell-dependent process (23, 24). Activation of autoreactive CD4+ T cells is induced by presentation of cryptic T cell epitopes that are not produced from a native Ag, rather than by an intrinsic abnormality of the immune system (25, 26). The proposed mechanisms that reveal cryptic T cell epitopes include structural modification or overexpression of autoantigens (27). In this regard, Zhou et al. (6) recently reported that some SSc-related autoantigens were overexpressed in SSc dermal fibroblasts compared with control dermal fibroblasts, suggesting that selective overexpression of SSc-related autoantigens in affected fibroblasts might induce expression of cryptic epitopes. A similar scenario can be proposed in the case of the anti-PHET Ab response, in which overexpression of PHET in SSc fibroblasts triggers the priming of specific T cells in genetically susceptible individuals with HLA class II alleles that potentially present PHET-derived cryptic peptides. This may explain the low frequency of anti-PHET Abs in SSc patients. The anti-PHET Ab response subsequently diversifies to the other portions of PHET and other variants by a process called an intramolecular epitope spreading (28). Alternatively, PHET overexpressed in the affected fibroblasts might be recognized as a non-self protein by the immune system, because the testis is one of the privileged sites that are sequestered from the immune system (29). In this regard, it is well known that a group of proteins called cancer-testis Ags, which are expressed exclusively in testis and cancer cells, are preferential targets of antitumor immune response (30). Taken together, dermal fibroblasts in the affected area of SSc patients may represent a cellular source of autoantigens that drives specific autoantibody responses.

The molecular function of PHET is currently unknown. One of the proteins in Hs.129872, c-Jun N-terminal kinase (JNK)-associated leucine zipper protein (JLP), was reported to be a member of the JNK-interacting proteins (31), which are scaffold proteins that form a complex with components of the JNK signaling pathway (32). A proposed function of the member of JNK-interacting proteins is promotion of effective phosphorylation of JNK through binding that facilitates its signal pathway (31). Although there is no report examining the role of the JNK signal in SSc fibroblasts, it is possible that overexpression of PHET and its variants in SSc dermal fibroblasts may augment the JNK signal pathway, resulting in enhanced proliferation and excessive production of extracellular matrix.

In summary, a testicular Ag PHET is a novel autoantigen recognized by sera from SSc patients with extensive fibrotic changes. The anti-PHET autoantibody response in SSc patients might be a result of up-regulated expression of PHET in dermal fibroblasts. Our findings should stimulate further studies examining a possible linkage between excessive fibrosis and specific autoantibody responses in SSc patients.


   Acknowledgments
 
We thank Carol A. Feghali and Tsuneyo Mimori for providing valuable comments, Kenichi Yamane for assisting with total RNA preparation, and Hidetoshi Inoko for assisting with nucleotide sequence analysis. The sequence data described have been submitted to the NCBI database under accession numbers AY219897 and AY219898.


   Footnotes
 
1 This work was supported by Keio University Medical Science Fund; a grant from the Ministry of Health, Labor, and Welfare; and a grant from the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Masataka Kuwana, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: kuwanam{at}sc.itc.keio.ac.jp Back

3 Abbreviations used in this paper: SSc, systemic sclerosis; JNK, c-Jun N-terminal kinase; MalBP, maltose-binding protein; PHET, protein highly expressed in testis; RNAP, RNA polymerase; RNP, ribonucleoprotein; rPHET, recombinant PHET; rPHET/Ex8-, recombinant PHET variant lacking exon 8; SLE, systemic lupus erythematosus; topo I, topoisomerase I. Back

Received for publication May 13, 2003. Accepted for publication October 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Medsger, T. A., Jr. 2001. Systemic sclerosis (scleroderma): clinical aspects. W. J. Koopman, Jr, ed. Arthritis and Allied Conditions 1590. Lippincott, Williams, and Wilkins, Philadelphia.
  2. Kuwana, M., J. Kaburaki, Y. Okano, T. Tojo, M. Homma. 1994. Clinical and prognostic associations based on serum antinuclear Abs in Japanese patients with systemic sclerosis. Arthritis Rheum. 37:75.[Medline]
  3. Okano, Y.. 1996. Antinuclear antibody in systemic sclerosis (scleroderma). Rheum. Dis. Clin. North Am. 22:709.[Medline]
  4. LeRoy, E. C.. 1992. A brief overview of the pathogenesis of scleroderma (systemic sclerosis). Ann. Rheum. Dis. 51:286.[Free Full Text]
  5. Korn, J. H.. 2001. Pathogenesis of systemic sclerosis. W. J. Koopman, Jr, ed. Arthritis and Allied Conditions 1643. Lippincott, Williams, and Wilkins, Philadelphia.
  6. Zhou, X., F. K. Tan, M. Xiong, D. M. Milewicz, C. A. Feghali, M. J. Fritzler, J. D. Reveille, F. C. Arnett. 2001. Systemic sclerosis (scleroderma): specific autoantigen genes are selectively overexpressed in scleroderma fibroblasts. J. Immunol. 167:7126.[Abstract/Free Full Text]
  7. Brentnall, T. J., D. Kenneally, A. J. Barnett, H. J. de Aizpurua, S. J. Lolait, R. Ashcroft, B. H. Toh. 1982. Autoantibodies to fibroblasts in scleroderma. J. Clin. Lab. Immunol. 8:9.[Medline]
  8. Hill, M. B., J. L. Phipps, R. J. Cartwright, A. Milford Ward, M. Greaves, P. Hughes. 1996. Antibodies to membranes of endothelial cells and fibroblasts in scleroderma. Clin. Exp. Immunol. 106:491.[Medline]
  9. Ronda, N., R. Gatti, R. Giacosa, E. Raschi, C. Testoni, P. L. Meroni, C. Buzio, G. Orlandini. 2002. Antifibroblast antibodies from systemic sclerosis patients are internalized by fibroblasts via a caveolin-linked pathway. Arthritis Rheum. 46:1595.[Medline]
  10. Chizzolini, C., E. Raschi, R. Rezzonico, C. Testoni, R. Mallone, A. Gabrielli, A. Facchini, N. Del Papa, M. O. Borghi, J. M. Dayer, et al 2002. Autoantibodies to fibroblasts induce a proadhesive and proinflammatory fibroblast phenotype in patients with systemic sclerosis. Arthritis Rheum. 46:1602.[Medline]
  11. Kuwana, M., K. Kimura, Y. Kawakami. 2002. Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera: application to enzyme-linked immunosorbent assay. Arthritis Rheum. 46:2742.[Medline]
  12. Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. 1980. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum. 23:581.[Medline]
  13. Tan, E. M., A. S. Cohen, J. F. Fries, A. T. Masi, D. J. McShane, N. F. Rothfield, J. G. Schaller, N. Talal, R. J. Winchester. 1982. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25:1271.[Medline]
  14. Medsger, T. A., Jr. 1996. Classification, prognosis. P. J. Clements, Jr, and D. E. Furst, Jr, eds. Systemic Sclerosis 51. Williams and Wilkins, Baltimore.
  15. Kuwana, M., J. Kaburaki, T. Mimori, T. Tojo, M. Homma. 1993. Autoantibody reactive with three classes of RNA polymerases in sera from patients with systemic sclerosis. J. Clin. Invest. 91:1339.
  16. Ihn, H., K. Tamaki. 2000. Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts: association with increased expression of the type I collagen gene. Arthritis Rheum. 43:2240.[Medline]
  17. Kuwana, M., J. Kaburaki, T. Mimori, T. Tojo, M. Homma. 1993. Autoantigenic epitopes on DNA topoisomerase I: clinical and immunogenetic associations in systemic sclerosis. Arthritis Rheum. 36:1406.[Medline]
  18. Heid, C. A., J. Stevens, K. J. Livak, P. M. Williams. 1996. Real time quantitative PCR. Genome Res. 6:986.[Abstract/Free Full Text]
  19. Kuwana, M., T. A. Medsger, Jr, T. M. Wright. 1995. Detection of anti-DNA topoisomerase I antibody by an enzyme-linked immunosorbent assay using overlapping recombinant polypeptides. Clin. Immunol. Immunopathol. 76:266.[Medline]
  20. Kuwana, M., Y. Okano, J. Kaburaki, T. A. Medsger, Jr, T. M. Wright. 1999. Autoantibodies to RNA polymerases recognize multiple subunits and demonstrate cross-reactivity with RNA polymerase complexes. Arthritis Rheum. 42:275.[Medline]
  21. Shankar, S., B. Mohapatra, A. Suri. 1998. Cloning of a novel human testis mRNA specifically expressed in testicular haploid germ cells, having unique palindromic sequences and encoding a leucine zipper motif. Biochem. Biophys. Res. Commun. 243:561.[Medline]
  22. Iyer, V. R., M. B. Eisen, D. T. Ross, G. Schuler, T. Moore, J. C. Lee, J. M. Trent, L. M. Staudt, J. Hudson, Jr, M. S. Boguski, et al 1999. The transcriptional program in the response of human fibroblasts to serum. Science 283:83.[Abstract/Free Full Text]
  23. Kuwana, M., T. A. Medsger, Jr, T. M. Wright. 1995. T and B cell collaboration is essential for the autoantibody response to DNA topoisomerase I in systemic sclerosis. J. Immunol. 155:2703.[Abstract]
  24. Kuwana, M., T. A. Medsger, Jr, T. M. Wright. 1995. T cell proliferative response induced by DNA topoisomerase I in patients with systemic sclerosis and healthy donors. J. Clin. Invest. 96:586.
  25. Kuwana, M., T. A. Medsger, Jr, T. M. Wright. 2000. Analysis of soluble and cell surface factors regulating anti-DNA topoisomerase I autoantibody production demonstrates synergy between Th1 and Th2 autoreactive T cells. J. Immunol. 164:6138.[Abstract/Free Full Text]
  26. Kuwana, M., C. A. Feghali, T. A. Medsger, Jr, T. M. Wright. 2001. Autoreactive T cells to topoisomerase I in monozygotic twins discordant for systemic sclerosis. Arthritis Rheum. 44:1654.[Medline]
  27. Lanzaveccia, A.. 1995. How can cryptic epitopes trigger autoimmunity?. J. Exp. Med. 181:1945.[Free Full Text]
  28. Fatenejad, S., M. J. Mamula, J. Craft. 1993. Role of intermolecular/intrastructural B- and T-cell determinants in the diversification of autoantibodies to ribonucleoprotein particles. Proc. Natl. Acad. Sci. USA 90:12010.[Abstract/Free Full Text]
  29. Filippini, A., A. Riccioli, F. Padula, P. Lauretti, A. D’Alessio, P. de Cesaris, L. Gandini, A. Lenzi, E. Ziparo. 2001. Control and impairment of immune privilege in the testis and in semen. Hum. Reprod. Update 7:444.[Abstract/Free Full Text]
  30. Boon, T., J. C. Cerottini, B. van den Eynde, P. van der Bruggen, A. van Pel. 1994. Tumor antigens recognized by T lymphocytes. Annu. Rev. Immunol. 12:337.[Medline]
  31. Kelkar, N., S. Gupta, M. Dickens, R. J. Davis. 2000. Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3. Mol. Cell. Biol. 20:1030.[Abstract/Free Full Text]
  32. Lee, C. M., D. Onesime, C. D. Reddy, N. Dhanasekaran, E. P. Reddy. 2002. JLP: a scaffolding protein that tethers JNK/p38MAPK signaling modules and transcription factors. Proc. Natl. Acad. Sci. USA 99:14189.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum ReprodHome page
R. Rana, N. Jagadish, M. Garg, D. Mishra, N. Dahiya, D. Chaurasiya, and A. Suri
Immunogenicity study of recombinant human sperm-associated antigen 9 in bonnet macaque (Macaca radiata)
Hum. Reprod., November 1, 2006; 21(11): 2894 - 2900.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yasuoka, H.
Right arrow Articles by Kuwana, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yasuoka, H.
Right arrow Articles by Kuwana, M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Nucleotide
*OMIM*Protein
*UniGene
Medline Plus Health Information
*Scleroderma

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS