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The Alloreactive T Cell Response Against the Class Ib Molecule H2-M3 Is Specific for High Affinity Peptides¹

Vikram M. Dabhi^*, Rolf Hovik^*,, Luc Van Kaer^, and Kirsten Fischer Lindahl^2,*,

^* Departments of Microbiology and Biochemistry, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75235; and Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232

	Abstract

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MHC class Ib molecule H2-M3 presents N-formylated peptides to CD8⁺ CTLs. Endogenous formylated peptides can come from the N-terminus of each of the 13 proteins encoded by the mitochondrial genome. In peptide competition assays, two of these peptides bind with high affinity, six bind with intermediate affinity, three bind with low affinity, and two do not bind measurably. Alloreactive CTLs from M3-specific, mixed lymphocyte cultures responded strongly against the two peptides with high affinity for M3, occasionally to peptides with intermediate affinity, and not at all to the rest. Long term lines and CTL clones reacted with only the high affinity peptides, demonstrating that alloreactive CTLs depend on specific peptides and that peptide affinity for class I correlates with alloantigenicity.

	Introduction

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Cytotoxic T lymphocytes recognize cell surface glycoproteins that consist of a polymorphic MHC class I heavy chain, an invariant ß₂m light chain, and a ligand, generally a peptide. During a normal immune response, CTLs target a specific peptide derived from a foreign protein complexed with a self class I molecule and lyse the presenting cell (1). CTLs also recognize and attack cells expressing non-self class I molecules, and these alloreactive CTLs mediate the rejection of class I-disparate tissue grafts. The peptide Ags involved have been difficult to identify, and two views of the ligand specificity of alloreactive T cells compete.

Matzinger and Bevan (57) proposed that alloreactive T cells recognize a complex of the foreign class I molecule and an associated cell-derived Ag. In normal cells, class I molecules are occupied exclusively with self peptides, and alloreactive CTLs may be specific for these peptide-MHC complexes. The following observations support this view. Alloreactive CTL clones lysed human cells expressing the mouse K^b class I molecule only when the cells had been treated with mouse cell lysate (2, 3). Different clones required different HPLC fractions from the lysate (4, 5). Some alloreactive CTLs recognize an allogeneic class I molecule only when the presenting cells are treated with an HPLC fraction derived from cells encoding a specific second MHC molecule (6). Only one such peptide has been identified: a leader peptide, derived from a class Ia molecule, H2-D or H2-L, and presented by Qa1, an MHC class Ib molecule (7). Non-MHC-derived peptides have also been implicated in allorecognition (6), but again only one peptide was identified; it is derived from the -ketoglutarate dehydrogenase protein and is associated with the class Ia molecule, H2-L^d (8).

In the other model, alloreactive T cells interact with determinants on a foreign class I molecule that are unaffected by bound peptide or the absence of peptide. These determinants would be present at a higher density than any determinant that requires a specific peptide complexed with the class I molecule (9). Because of the compensatory high ligand density, T cells, even with low affinity recognition, would efficiently kill these target cells (10). The following examples support that model. Isolated HLA-A2 molecules, reconstituted in the absence of peptide and therefore empty, stimulated an IL-2-dependent proliferative response by HLA-A2-specific alloreactive T cells (11). These putatively peptide-free HLA-A2 molecules induced a response comparable to a preparation of native HLA-A2 molecules, not subjected to denaturation and renaturation, suggesting that some of these are also unoccupied. It was also found that some alloreactive CTLs lysed the TAP2 mutant RMA-S cells incubated at 26°C, but not at 37°C (12). At the lower temperature, presumably empty class I molecules that express these alloepitopes are stably expressed on the cell surface. The empty class I model and the Matzinger and Bevan model are not mutually exclusive, as an alloresponse could generate two kinds of CTLs, i.e., CTLs that recognize either peptide-dependent or independent epitopes.

We studied alloreactivity using a class Ib molecule, H2-M3, that presents endogenous peptides as minor histocompatibility Ags to CD8⁺ CTLs (13), but differs from other class I molecules by its strong preference for peptides that are formylated at the N-terminus (14, 15). In a normal cell, N-formylated peptides can come only from the N-terminus of mitochondrially encoded proteins, limiting to 13 the number of endogenous peptides that can be presented. One of these mitochondrial proteins, ND1, a subunit of NADH dehydrogenase, is polymorphic at the sixth residue among mouse strains; the alleles are = 6I, ß = 6A, = 6V, = 6T (16). This difference is detected by CTLs that recognize the ND1-derived formylated peptide presented by H2-M3 (17). N-formylated peptides can also originate from bacterial proteins during an infection; indeed, M3 participates in the immune response against Listeria monocytogenes (18, 19, 20).

MHC class I alleles usually differ at residues that line the peptide binding groove. These differences determine which peptides bind to a given allele and what conformation that peptide will take. M3, like other class Ib molecules, shows little polymorphism; only six allelic forms have been identified, differing by a maximum of eight residues in the 1 and 2 domains. Two of these allelic forms, M3^wt and M3^cas, differ at positions 31 (Val to Met) and 95 (Leu to Gln) (17). The side chain of residue 31 points toward the 3 domain and is not directly involved in peptide binding. The side chain of residue 95 points up into the peptide groove and interacts directly with the side chain of the third and sixth residues of the peptide (21). The wild-type molecule, M3^wt, expressed by most common laboratory strains, presents all four ND1 peptides and Listeria peptide fractions. We challenged M3^cas mice with cells from an H2-compatible, M3^wt mouse and took advantage of the narrow range of endogenous peptides that M3 can bind to determine what the responding, alloreactive, M3-specific CTLs see. Our findings show not only that specific peptides are required for allorecognition, but also that there is a direct correlation between a peptide’s affinity for the class I molecule and its ability to provoke an alloresponse.

	Materials and Methods

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Mice and nomenclature

C57BL/6J (B6), C3H/HeJ, and B10.BR/SgSnJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and BALB.B mice were purchased from Harlan (Bicester, Oxon, U.K.). The TAP1^o and TAP1⁺ littermates on a 129 strain background (M3^wt) (22) were bred at University of Texas Southwestern Medical Center by J. Forman. Because of the limited availability of TAP1⁺ littermate mice, lymphoblasts were stored frozen and recultured in SMDM-IL2 medium (23) for 36 h before assay. The following strains were bred in our colony: BALB.B.B2m^w5 (24, 25), B6-mt^WLA (26), B6.CAS3(R9), and B6.CAS3(R4) (27). The recombinant B6.CAS3(R9) carries a haplotype with H2-K through H2-D from B6 and the rest of H2 from Mus musculus castaneus (cas3), including M3, whereas recombinant strain B6.CAS3(R4) carries a chromosome with most of H2 (H2-K through H2-T1) from strain C3H/HeJ, but M3 from cas3 (28). Both were made homozygous on the B6 genetic background, R4 at N2 and R9 at N10.

Mta (for maternally transmitted Ag) describes the ND1-derived peptide complexed with M3. To describe the Mta phenotype of our mouse strains and cell lines, we annotate them with square brackets giving the mt-ND1 (formerly Mtf) allele followed by the M3 allele (formerly Hmt). For example, C57BL/6J [6I,wt] describes the C57BL/6J strain as mt-ND1^6I and M3^wt.

The strain combinations used to raise CTLs are listed in Table I. To generate the alloreactive M3^cas-anti-M3^wt CTLs, we immunized B6.CAS3(R4) [6I,cas] mice with C3H [6I,wt] spleen cells to evoke a response against M3^wt. The minor C3H Ags provide help in vivo. In vitro stimulation with B10.BR [6I,wt] focused the activation to those CTLs responding against M3^wt. All three strains carry ND1^6I mitochondria, and we thus avoid responses against mitochondrial peptide differences.

MLCs, CTL lines, and clones

MLCs and CTL lines were generated in the strain combinations defined in Table I, cloned by limiting dilution, and maintained as previously described (23). The anti-Qa1^b clone, provided by C. Aldrich and J. Forman, was generated from a B6.Tla^a (H2^b, Qa1^a)-anti-B6 (H2^b, Qa1^b) MLC (30, 31).

Media

The following media were used: complete RPMI 1640, RPMI 1640 supplemented to final concentrations of 10 mM HEPES, 4 mM L-glutamine, 50 µM 2-ME, 100 IU/ml penicillin, and 100 µg/ml streptomycin; and RPMI-10 and RPMI-2, complete RPMI 1640 supplemented with 10 or 2% (v/v) FCS that has been heat-inactivated at 56°C for 30 min.

Peptides

Peptides were synthesized on a Rainin Symphony peptide synthesizer (Rainin, Woburn, MA), using F-moc amino acids under the standard manufacturer’s conditions and were analyzed as previously described (15). They were nine amino acids long, starting from the N-terminus of each of the 13 mitochondrial proteins (Table II) and including ND1-6A, ND1-6T, ND1-6V, and COI-3T (fMFTNRWLFS); ND2.4187 was an internal peptide of ND2 from residues 92–100 (Table II). All peptides were dissolved in DMSO at stock concentrations of 0.2 µM to 20 mM.

Standard CTL assays were conducted in RPMI-10. For target cells we used transformed cell lines, RMA (from strain C57BL/6; [6I,wt] H2^b) (32), RMA-S (32, 33), or Pc11198 (from strain NZB/Icr; [6A,wt] H2^d2) (34), maintained in logarithmic growth phase, and spleen cells that were stimulated with Con A for 48 h (lymphoblasts) (23). For target cells incubated with peptide, each figure or table gives the incubation time and concentration. Target cells were washed free of excess peptide, labeled with ⁵¹Cr for 1 h, and washed twice with RPMI-2. Standard ⁵¹Cr release assay protocols were followed as previously described (23). The percentage of specific ⁵¹Cr release was calculated as 100 x (experimental release - spontaneous release)/(maximum release). Spontaneous release varied between 4–25% of maximum release. The error was <5% in all experiments.

Best-fit curves and IC₅₀

The SlideWrite Plus for Windows 3.0 program from Advanced Graphics Software (Carlsbad, CA) was used to derive best-fit curves for the peptide inhibition data. A nonlinear regression fit to a scatter plot was made with the dose-response logistics equation: y = a₀ + a₁/[1 + (x/a₂)^a3]. From this equation, the concentration (x) of competitor peptide required to inhibit killing (y) by 50% (IC₅₀) was calculated.

	Results

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Alloreactive response specific for M3

The alloreactive killers, from recombinant M3^cas strain B6.CAS3(R4) immunized with H2^k-matched, M3^wt cells (Table I), were indeed specific for M3^wt. For example, the alloreactive long term line, LT-0701C,² failed to lyse B10.CAS2 fibroblasts, which carry the M3^cas allele (35). When these fibroblasts were stably transfected with M3^wt cDNA, the resulting CM3 cells were lysed by CTL line LT-0701C (Fig. 1) (15). In addition, LT-0701C lysed lymphoblasts from M3^wt strains C57BL/6 and BALB/c, but not from recombinant M3^cas strains (B6.CAS3(R9) and B6.CAS3(R4); Fig. 1 and data not shown); this ruled out that lysis of H2^b (B6) or H2^bc (TAP1⁺) target cells was due to a cross-reaction against MHC class Ia, which they share with R9. All the lines and clones tested reacted similarly against the fibroblasts and the recombinant strains.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Alloreactive CTLs specifically recognize M3wt. The alloreactive line LT-0701C was tested for cytolytic activity against B10.CAS2 ([6I,cas] H2w17) fibroblasts (), CM3 B10.CAS2 fibroblasts transfected with M3wt cDNA (•), and B6.CAS3(R9) (B6.R9, M3cas) () and C57BL/6 (B6, M3wt) () lymphoblasts at highest the E:T cell ratio.

TAP1 and TAP2 form an ATP-dependent transporter that is required for moving peptides into the endoplasmic reticulum (36). To test whether the alloreactive CTLs recognize an Ag that depends on peptides for surface expression, we used lymphoblasts from TAP1^o mice (22) and TAP2-deficient RMA-S cells. Both express low levels of class I molecules and were not killed by line LT-0701C (Fig. 2). Lymphoblasts from TAP1⁺ littermate mice and TAP2⁺ RMA cells were lysed, demonstrating that the CTLs were capable of lysing targets and recognized Ags that required both TAP molecules for cell surface expression. All alloreactive lines and clones showed this property of much weaker or no reactivity with untreated TAP-deficient target cells compared with wild-type cells (as shown below).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. M3 alloantigen recognition requires TAP1 and TAP2 expression. Alloreactive line LT-0701C was tested for cytolytic activity against lymphoblasts from TAP1o () and TAP1+ littermate mice (•) and against TAP2 mutant RMA-S () and wild-type RMA control () cells.

The mouse mitochondrial genome encodes 13 proteins; their synthesis can be specifically inhibited by chloramphenicol. To determine whether any of these proteins are required for allorecognition, we treated lymphoblast targets with various concentrations of chloramphenicol for 24 h. A Qa1^b-specific CTL clone killed untreated and chloramphenicol-treated target cells equally well (Fig. 3) because it recognizes the class Ib molecule Qa1 with a nuclearly encoded peptide. Killing of the lymphoblasts by the M3-specific line LT-1017 decreased with increasing concentrations of chloramphenicol, suggesting that the Ag recognized by these CTLs has a mitochondrial peptide component.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Chloramphenicol inhibits M3-specific allorecognition of target cells. M3cas-anti-M3wt line LT-1017 (; E:T cell ratio, 2:1) and an alloreactive anti-Qa1b clone (; E:T cell ratio, 5:1) were tested on B6-mtWLA lymphoblasts ([6V,wt] Qa1b) treated with the indicated concentrations of chloramphenicol for 24 h. Free-standing symbols represent lysis of untreated cells.

Naturally processed, formylated peptides from the N-terminus of any of the 13 mitochondrial proteins might be part of an M3 alloantigen. We tested the ability of 13 corresponding synthetic, 9-mer peptides to compete with the ND1-6V peptide for binding to M3^wt on Pc11198 target cells. These cells express the ND1-6A peptide, which the -anti- line and clone CE9 do not recognize; Pc11198 cells are therefore not killed unless sensitized with ND1-6V peptide (Table II). The first competitor, ND1-6I, should, and did, block lysis because it differs from ND1-6V only at the sixth residue (Ile, Val) and is known to bind M3 (15, 16). Seven other competitor peptides prevented killing by both the -anti- line and clone CE9; three additional peptides, ATPase 6, ATPase 8, and ND4L, inhibited killing by CE9, but did not inhibit killing by the CTL line, which presumably has higher affinity for its target Ag; two peptides, COIII and CYTb, failed to block lysis by either the line or the clone or by two additional ND1-6V-specific lines (data not shown). The unformylated peptide ND2-4187 served as a negative control and did not prevent killing. Thus, 11 of the mitochondrial N-terminal peptides can bind M3 detectably and are potentially part of an M3 alloantigen.

To determine the relative binding affinity of these peptides, mt-ND1^6A target cells were incubated with a limiting dose of ND1-6V and various concentrations of the competing peptides and were then tested for recognition by an -anti- line. For each competitor peptide, we made a scatterplot of the inhibition data and derived best-fit curves (Fig. 4), which provided the inhibitor concentration that produced half-maximal killing (IC₅₀; Table II). The peptides could be classified into four groups: the ND1 and COI peptides compete at the lowest concentrations (1–100 nM); the ND2, ND3, ND4, ND5, ND6, and COII peptides compete in the 0.1–2 µM range; three peptides, ATPase6, ATPase8, and ND4L, compete in the 3–12 µM range; and two peptides, COIII and CYTb, failed to compete in the concentration range tested (data not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Formylated mitochondrial peptides inhibit lysis of cells treated with the ND1-6V 9-mer. -Anti- CTLs were tested for killing of Pc11198 ([6A,wt] H2d) target cells incubated with ND1-6V 9-mer at a final concentration of 7.5 nM, and the competitor peptide at the indicated concentrations. Each curve represents a separate experiment (E:T cell ratios, 1:1 to 12:1).

Mitochondrial peptides recognized by MLCs

An alloreactive MLC established with the B6.CAS3(R4)-anti-C3H/B10.BR strain combination contains a multitude of CTLs and thus reflects an in vivo response. The bulk-cultured CTLs were tested for peptide specificity with lymphoblast targets (from TAP1^o mice) incubated with each of the 11 peptides that competed for binding and with the noncompetitive peptide CYTb. The CTLs lysed cells treated with the ND1 and the COI peptides at a level comparable to TAP1⁺ littermate cells and ND3 peptide-treated cells at lower levels (Fig. 5). Those peptides not shown in the figure failed to sensitize the lymphoblasts (Table III). The CYTb peptide served as a negative control.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. M3wt-specific, alloreactive cultures recognize mitochondrial peptides ND1, COI, and ND3. CTLs from 5-day bulk MLCs were tested for cytolytic activity against TAP1o lymphoblasts incubated without peptide or with the indicated mitochondrial peptides overnight at a final concentration of 2 µM. Lymphoblasts from a TAP1+ littermate mouse were assayed without peptide treatment.

Mitochondrial peptides recognized by alloreactive clones and lines

Long term CTL lines (LT) were established from some of the MLCs, and clones were isolated from a few of these lines (Table I); these killers were tested to better define their alloantigen specificity. RMA-S cells and lymphoblasts from TAP1^o mice were incubated with each of the 11 formylated mitochondrial peptides that bind to M3. The COI peptide sensitized targets to lysis by four clones and two lines (Table IV); the ND1-6I peptide sensitized targets to clone EC6 and four long term lines; clone EC6 also killed targets incubated with ND3, but less well, mimicking the recognition pattern of the MLC in Fig. 5.

M3-restricted CTL response to ND1 and COI peptide alleles

Alleles of the COI and ND1 peptides, which differ by a single residue, were tested to determine whether the changes affect allorecognition. The sixth residue of the ND1 protein is polymorphic (16); synthetic peptides ND1-6I, -6A, -6V, and -6T sensitized lymphoblasts from TAP1^o mice to lysis by ND1-dependent, alloreactive lines LT-4, -6, and -7 at comparable levels (Table V). Thus, CTLs in these lines are insensitive to conformational changes induced by the buried sixth residue. The control -anti- line lysed only target cells sensitized with ND1-6I, demonstrating that changes at this position affect peptide-specific CTL recognition.

	Discussion

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Bevan (10) proposed that the TCR recognizes conformations that differentiate allogeneic class I from self-class I molecules and that peptide-class I complexes expressed at a high level would trigger lysis by alloreactive CTL. Although formylated peptides from either ND1 or COI both induce surface display of M3 alloantigen on RMA-S cells and on lymphoblasts from TAP1^o mice, all the long term lines and clones, except EC6, responded to only one of the two peptides (Table IV). For example, clones specific for COI did not lyse target cells treated with the ND1 peptide, which binds with a higher affinity than the COI peptide and induces cell surface expression of ND1-M3 complexes (Table IV). Therefore, many alloreactive CTLs discriminate among different peptides complexed with the same class I molecule and show that peptide association results in epitope specificity (38).

Clone EC6 lysed cells treated with the COI peptide best, but also reacted with target cells treated with ND1 or ND3 peptides (Table IV); as determined by sequencing (39), EC6 has a single productive TCR ß- and -chain, which therefore must cross-react with three different mitochondrial peptides, each complexed to M3. Thus, EC6 represents an alloreactive CTL that is peptide dependent but not specific.

Bulk cultures and some M3-reactive clones (e.g., DC4) lysed RMA-S cells and lymphoblasts from TAP1^o not treated with peptide (Tables III and IV). Similarly, CTL alloreactive against other class I molecules, such as K^b, lysed TAP-deficient cells not incubated with peptide (12). These target cells express on their surface a low density of class I molecules detectable with mAbs (40, 41, 42). Proteins resident in the ER and in the medium can contribute peptides that stabilize empty class I molecules (43). Potent CTLs with high affinity receptors may sense the few peptide-class I complexes and lyse the cell. When the target cells were treated with the appropriate peptide (12), lytic activity increased two- to threefold, demonstrating recognition mediated by peptides. Similar observations were made for the M3-reactive clones and bulk cultures that lysed TAP-deficient cells (Tables III and IV), indicating that recognition of M3 by these CTL is also peptide specific or dependent.

Sequence sensitivity is context dependent

Small changes in the peptide can alter the overall conformation of the peptide-class I complex and result in the loss or gain of serologic epitopes (44). T cell reactivity will depend on which epitopes are lost and how critical they are for recognition in a given context. For example, the M3-alloreactive CTLs were insensitive to changes at the sixth residue of ND1 (Ile, Ala, Val, or Thr; Table V), whereas peptide-specific, M3-restricted CTLs differentiate among the ND1 alleles (Table V) (15, 16, 28). In crystals of M3-ND1, the side chain of P6 points into the groove (21), making direct interaction with the TCR unlikely; however, the different ND1 peptides do change the M3-peptide complex, such that it acts as a minor histocompatibility Ag. By contrast, a change at P3 of the COI peptide affects both alloreactive and peptide-specific CTL; the complex of M3 with the P3-Thr peptide is poorly recognized by alloreactive clones DC4 and EC6 (Table VI), whereas P3-Ile or P3-Val is recognized by M3-restricted CTL from P3-Thr mice. A crystal structure of the COI peptide-M3 complex is not available, but it is likely that the P3 side chain assumes a position similar to that in ND1, where it points sideways toward the 1 helix and is potentially accessible to a TCR (21).

M3 binding and immunodominance

The interactions formed between the binding groove and side chain residues define the sequence selectivity of a particular class I molecule (45). An N-formyl group on a peptide increases the binding affinity for M3 (15, 46), but key residues at other positions also contribute to and determine the overall affinity, as has been reviewed and discussed previously (28, 47).

All the alloreactive M3^cas-anti-M3^wt CTLs reacted with TAP-negative target cells treated with mitochondrial formylated peptides from the N-terminus of either the ND1 or COI polypeptide. Of the 13 formylated mitochondrial peptides, these two (ND1-6I and COI-3I) bound with the highest affinity as determined by peptide competition. Similarly, in the Qa1 class Ib system the alloreactive response is dominated by a single Qdm peptide (7), and an alloreactive response against L^d requires only the 2C peptide (8). Both cases may involve high affinity binding by each class I molecule of its respective peptide. Even in a self-restricted response, for example against OVA, CTLs react predominantly to K^b-OVA_257–264 complexes (48, 49); OVA contains at least five other epitopes with K^b binding motifs (50), but the peptide that corresponds to OVA_257–264 binds best (51). Thus, peptides with high affinity for a class I molecule can provoke a strong CTL response in both self-MHC-restricted and alloreactive T responses (52, 53).

One should still ask why of the many M3-alloreactive CTLs so few react against M3 complexed with ND2, ND4, COII, ND3, ND5, and ND6, because these bind with an affinity not much lower than that of COI. In a systematic study of the class I molecule, K^d, Deng and co-workers (54) found that peptide binding affinity, although important, is not the sole factor that determines the CTL response; peptide liberation, TAP-mediated peptide transport, and T cell repertoire can all limit immunogenicity. In addition to these factors, the stability of the peptide-MHC complex, as measured by the dissociation rate, has turned out to be a major predictor of immunogenicity (55, 56).

	Acknowledgments

 
We thank Clive Slaughter, Lynn Mayfield, and Bikash Pramanik for peptide synthesis and analysis; James Forman for providing TAP1^o mice; Margaretan Stolfo, Henry Taylor, and Anne Eubanks for care and breeding of the mice; and Chyung-Ru Wang and Derek Byers for discussions.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Kirsten Fischer Lindahl, Howard Hughes Medical Institute, 5323 Harry Hines Blvd., Dallas, TX 75235-9050. E-mail address:

² Abbreviations used in this paper: LT, long term lines; cas, castaneus.

Received for publication May 5, 1998. Accepted for publication July 7, 1998.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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