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The Journal of Immunology, 1999, 163: 1763-1768.
Copyright © 1999 by The American Association of Immunologists

Strain Variation in Autoimmunity: Attempted Tolerization of DA Rats Results in the Induction of Experimental Autoimmune Encephalomyelitis1

Derek C. Lenz, Norbert A. Wolf and Robert H. Swanborg2

Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This paper reports that DA rats develop experimental autoimmune encephalomyelitis (EAE) when immunized with encephalitogenic myelin basic protein (MBP) peptide (MBP63–81) in IFA. In contrast, most rodent strains are tolerized by this procedure. Doses as low as 5 µg peptide + IFA induced EAE in DA rats. Lewis (LEW) rats did not develop EAE, even after immunization with 100 µg encephalitogenic peptide (MBP68–86) + IFA, but were rendered tolerant to EAE. DA rat T cells proliferated to peptide, and proliferation was inhibited by CTLA4Ig, and by anti-B7.1 and anti-B7.2 mAbs. This indicates that the ease of induction of EAE in this strain does not reflect a decreased requirement for T cell costimulation through the B7/CD28 costimulatory pathway. The inhibitory effect of CTLA4Ig was abrogated in the presence of anti-TGF-ß-neutralizing Ab. An encephalitogenic DA T cell line expressed mRNA for the Th1 cytokines IFN-{gamma} and TNF-{alpha}, as well as IL-10, and secreted these cytokines. In contrast, a T cell line from peptide + IFA-immunized LEW rats (which did not develop EAE) failed to secrete these cytokines. Although this line did not express TNF-{alpha} or IL-10 mRNA, IFN-{gamma} mRNA was detected, suggesting posttranscriptional regulation of IFN-{gamma} expression. Attempts to induce unresponsiveness in DA rats with encephalitogenic peptide-coupled splenocytes were also unsuccessful.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE)3 is a Th1 inflammatory response directed at the CNS of susceptible laboratory animals (1). The autoreactive CD4+ T cells cross the blood-brain barrier and secrete the proinflammatory cytokines IFN-{gamma} and TNF-{alpha} in response to epitopes present on CNS proteins, including myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein.

Although there have been sporadic reports that EAE can be induced in rats without CFA, those studies have generally required the use of crude spinal cord homogenates in IFA to elicit the disease (2, 3). These homogenates may contain adjuvant-like components.

The more general finding has been that injections of encephalitogenic Ag in IFA render animals tolerant with respect to EAE (4, 5, 6, 7). It has been proposed that suppressor T cells (Ts) (6, 7), immune deviation from a Th1 to a Th2 immune response (8) (9), or anergy (10) may account for this unresponsive state. A recent report also suggests that soluble MBP treatment of Lewis (LEW) rats with EAE increases Fas-mediated apoptosis of autoreactive T cells in the CNS that coincides with amelioration of the disease (11).

Another effective method of inducing immunological unresponsiveness is the i.v. injection of splenocytes coupled with protein, hapten, or peptide (12). This typically results in a profound state of tolerance to the moiety conjugated to the injected splenocytes and has been effectively utilized to inhibit EAE in rats (13).

In the present report, we immunized DA rats with a major DA-specific encephalitogenic MBP epitope, MBP63–81 (14) and LEW rats with the dominant LEW-specific epitope, MBP68–86 (15, 16) administered in IFA. We evaluated EAE, as well as T cell proliferative responses and cytokine profiles in the respective strains. In addition, we attempted to tolerize LEW and DA rats with peptide-coupled splenocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides

Synthetic peptides were prepared using F-moc chemistry with an Applied Biosystems Synergy model 432A peptide synthesizer (Perkin-Elmer, Foster City, CA), according to the manufacturer’s instructions, and structure was confirmed by electrospray mass spectrometry, as previously described (14). Purity of most peptides exceeded 95%. The peptides were numbered according to the bovine MBP sequence (17). The peptides used in these experiments were MBP63–81 (ARTTHYGSLPQKSQRSQ), which is a major encephalitogenic epitope for DA rats, MBP68–86 (YGSLPQKSQRSQDENPV), which is the dominant encephalitogenic epitope for LEW rats, and MBP73–86 (QKSQRSQDENPV), which is the core LEW epitope.

Animals and immunization

Eight- to 12-wk-old DA and LEW rats (purchased from Harlan Sprague Dawley, Indianapolis, IN, and Charles River, Raleigh, NC, respectively) were immunized s.c. with the appropriate synthetic MBP peptide, emulsified in IFA (Difco, Detroit, MI). Both male and female DA rats were evaluated; no gender-related differences were noted. They were observed for clinical signs of EAE, graded as 0 (no disease), 1 (loss of tail tonicity), 2 (hind limb weakness), or 3 (hind limb paralysis), as previously described (18). In some experiments, rats were immunized with peptide in CFA (see text). Optimal doses for ensuring induction of paralytic EAE were: 2.5 µg MBP73–86 or MBP68–86 + CFA in LEW rats, and 10 µg MBP63–81 + CFA in DA rats. Hematoxylin-eosin-stained spinal cord sections from representative rats were examined microscopically for inflammatory infiltrates without knowledge of the group of origin.

T cell proliferation

The T cell proliferation assay was performed as previously described (13, 18). Briefly, splenocytes were isolated from peptide-primed rats, adherent cells were removed by culture on plastic petri dishes, and T cells were isolated on T cell columns (Biotec, Edmonton, Canada). The T cells were cultured for 96 h with irradiated (2000 rad) syngeneic thymocytes as APCs, and peptide, in 96-well flat-bottom microtiter plates. The cultures were pulsed with [3H]thymidine (0.5 µCi/well) 18 h before harvesting the cells, and [3H]thymidine incorporation was measured in a Microbeta Plus liquid scintillation counter (Wallac, Gaithersburg, MD). Cultures were run in triplicate or quadruplicate, and each experiment was repeated at least three times. Dose-response studies were performed using various peptides at differing concentrations, and representative results are presented. The stimulation index (S.I.) was calculated as cpm with peptide/background (cpm of T cells and APCs without peptide). SI was considered significant only if it exceeded background by at least 3-fold.

To evaluate costimulatory requirements, various concentrations of CTLA4Ig (R&D Systems, Minneapolis, MN), and rat anti-B7.1 or anti-B7.2 mAbs (clones 3H5 and 24F, respectively, PharMingen, San Diego, CA) were added to microtiter wells in proliferation assays. Turkey anti-human TGF-ß Abs that neutralize rat TGF-ß (Collaborative Biomedical Products, Bedford, MA) were used to ascertain whether TGF-ß plays a role in CTLA4Ig-induced inhibition of rat T cell proliferative responses.

T cell lines

T cell lines were generated from spleens of DA and LEW rats immunized with the respective strain-dominant encephalitogenic MBP peptides, as previously described (18). The lines were expanded alternately in Con A supernatant containing 25 U/ml IL-2, or with peptide containing medium. MBP63–81 and MBP68–86 were used to select peptide-specific DA and LEW T cells, respectively.

RNA isolation

Total cellular RNA was isolated from T cell lines using commercial RNA isolation kits (Qiagen, Santa Clarita, CA). The RNA was treated with RNase-free DNase (Promega, Madison, WI) before reverse transcription. The purity of the RNA was determined by comparing OD260/OD280 using an Ultraspec III spectrophotometer (Pharmacia, Piscataway, NJ). An aliquot of each product was run on a 1.9% agarose gel in 1x TAE buffer and examined for the presence of 18S and 28S bands to confirm integrity of the RNA.

RT-PCR

sscDNA was synthesized from the total RNA by reverse transcription using oligo-dT primer and AMV reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, as previously described (19). PCR was performed on aliquots of RT products using rat-specific PCR primers for IFN-{gamma}, TNF-{alpha}, and IL-10; ß-actin primers were used as "housekeeping" gene controls (Clontech, Palo Alto, CA; and Biosource International, Camarillo, CA).

PCR products were separated on 1.9% agarose gels in 1x TBE buffer containing 5 µg/ml ethidium bromide. Product sizes for cytokines were 288 bp for IFN-{gamma}, 295 bp for TNF-{alpha}, and 376 bp for IL-10. The product size for ß-actin was 457 bp.

Evaluation of secreted cytokines

Supernatants from peptide-cultured T cell lines were assayed for IFN-{gamma}, TNF-{alpha}, and IL-10 using commercial ELISA kits (Life Technologies, Grand Island, NY; and Biosource International).

Tolerance induction with peptide-coupled splenocytes

Encephalitogenic peptide was coupled to rat splenocytes using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (ECDI, purchased from Calbiochem, La Jolla, CA), as described by Malotky et al. (13). We prepared MBP68–86-ECDI-splenocytes and "sham" ECDI-splenocytes from LEW rats, and MBP63–81-ECDI-splenocytes and "sham" ECDI-splenocytes from DA rats (13). These were injected i.v. into groups of LEW and DA rats, respectively (5 x 107/rat), and the rats were challenged 4 days later with the respective peptide in CFA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We previously reported that MBP63–81 is a major encephalitogenic epitope for DA rats when administered in CFA (14). With the intent of inducing tolerance, we immunized four DA rats with 50 µg of MBP63–81 in IFA. The purity of this peptide exceeded 95%. Unexpectedly, all four DA rats developed clinical EAE 9 days postimmunization. Table IGo summarizes the results of eleven experiments. As shown, 54 of 55 (98%) DA rats immunized with various doses of MBP63–81 in IFA developed clinical EAE. A dose as low as 5 µg was encephalitogenic. In contrast, LEW rats did not develop EAE when immunized with MBP68–86 + IFA (0/30, Table IGo). Rather, this treatment elicited unresponsiveness as confirmed by failure to develop clinical EAE when subsequently challenged with MBP68–86 + CFA (results not shown).


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  		Table I. Induction of EAE in DA but not LEW rats with encephalitogenic peptide + IFA

 
In confirmation of the clinical status, extensive perivascular and parenchymal mononuclear cell inflammation was observed in the spinal cords of DA rats with EAE (Fig. 1Go). In contrast, spinal cord sections of LEW rats that received MBP68–86 + IFA were usually negative, although rare isolated perivascular cuffs were occasionally seen.



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  		FIGURE 1. Hematoxylin-eosin stained section of spinal cords from DA rats immunized with MBP63–81 + IFA, showing extensive mononuclear cell infiltration. A, x60; B, x120. Both rats had clinical EAE.

 
In vitro recall responses to the priming peptide were evaluated by T cell proliferation assays. As shown in Fig. 2Go, T cells from MBP63–81-primed DA rats responded vigorously to the immunizing peptide (5 µM). In contrast, T cells derived from LEW rats immunized with MBP68–86 + IFA responded poorly to the priming peptide. Similar results were obtained when the T cells were stimulated with 20 µM peptide. Moreover, 5 µM MBP68–86 stimulates robust in vitro responses in T cells from MBP68–86 + CFA-primed LEW rats with EAE (results not shown).



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  		FIGURE 2. Proliferative responses of T cells to the priming peptide: DA rats immunized with MBP63–81 + IFA, and LEW rats immunized with MBP68–86 + IFA.

 
The activation of CD4+ T cells requires two signals, one delivered by peptide + MHC class II to the TCR, and the second, a costimulatory signal, delivered through ligation of CD28 on the CD4+ T cell and B7-1 or B7-2 on the APC (20). It has been shown that blockade of the CD28/B7 pathway inhibits acute and chronic relapsing EAE (21, 22, 23, 24), and prevents epitope spreading in mice (22). One possible explanation for the unique susceptibility of DA rats to EAE is that costimulatory requirements for the activation of self-reactive T cells are less stringent in this strain.

To test this hypothesis, we attempted to block T cell proliferative responses of MBP-63–81 + IFA-primed DA rats with various concentrations of CTLA4Ig, as well as with anti-B7.1 and anti-B7.2 mAbs. We also immunized LEW rats with MBP73–86 + CFA to induce EAE, and cultured those encephalitogenic T cells with APCs and MBP73–86, with or without CTLA4Ig, or anti-B7.1, or anti-B7.2. As shown in Fig. 3Go, CTLA4Ig significantly inhibited the proliferation of T cells from LEW rats immunized with MBP73–86 + CFA, and paralyzed with EAE. The anti-B7.1 and anti-B7.2 mAbs also inhibited proliferation. CTLA4Ig, anti-B7.1, and anti-B7.2 also inhibited the proliferative response of T cells from paralyzed DA rats immunized with MBP63–81 + IFA (Fig. 4Go). We did not observe a differential effect of anti-B7.1 vs anti-B7.2 in either the LEW or DA responses. These findings indicate that B7/CD28 costimulation is required in both strains.



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  		FIGURE 3. B7/CD28 costimulation is required for proliferative response of MBP73–86 + CFA-primed LEW rats with EAE to the priming peptide. *, p < 0.05 vs peptide alone; **, p < 0.02 vs peptide alone.

 


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  		FIGURE 4. B7/CD28 costimulation is required for proliferative response of MBP63–81 + IFA-primed DA rats with EAE to the priming peptide. *, p < 0.01 vs peptide alone; **, p < 0.001 vs peptide alone.

 
It was recently reported that CTLA-4 engagement leads to production of TGF-ß by murine CD4+ T cells, which contributes to the down-regulation of T cell activation (25). Thus, we sought to determine whether TGF-ß plays a role in CTLA4Ig-mediated inhibition of rat T cell proliferative responses. We immunized DA rats with MBP63–81 + IFA, and LEW rats with MBP73–86 + CFA for induction of EAE. T cell proliferation assays were conducted as described above, except that neutralizing anti-TGF-ß Ab was added to some microtiter wells. The results in Fig. 5Go confirm that CTLA4Ig inhibits the LEW and DA T cell responses to the respective priming peptides, and also reveal that anti-TGF-ß Ab substantially restores these responses. This suggests that TGF-ß is involved in CTLA4Ig-induced inhibition of rat T cell responses.



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  		FIGURE 5. Anti-TGF-ß abrogates CTLA4Ig-induced inhibition of T cell proliferative responses. A, DA rat proliferative response to MBP63–81; B, LEW rat proliferative response to MBP73–86. *, p < 0.001 CTLA4Ig + anti-TGF-ß vs CTLA4Ig alone.

 
To test the hypothesis that the disparate responses of peptide + IFA-immunized LEW and DA rats reflects differential patterns of cytokine production, we generated T cell lines from each strain. T cell line DA/IFA was prepared from a DA rat immunized with MBP63–81 + IFA; this rat developed EAE. T cell line LEW/IFA was obtained from a LEW rat that had been immunized with MBP73–86 + IFA; this rat did not develop EAE and was presumably tolerant. As shown in Fig. 6Go, T cell line DA/IFA secreted ~2000 pg TNF-{alpha} and ~600 ng IFN-{gamma} (note that scales are in pg and ng, respectively). The DA/IFA line also secreted IL-10. These cytokines were not secreted by T cell line LEW/IFA.



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  		FIGURE 6. Cytokine production by T cell lines derived from DA and LEW rats (lines DA/IFA and LEW/IFA, respectively) immunized with peptide in IFA.

 
As expected, IFN-{gamma}, TNF-{alpha}, and IL-10 mRNA was expressed by the encephalitogenic DA/IFA cell line (Fig. 7Go, lane 1). In contrast, T cell line LEW/IFA did not express TNF-{alpha} or IL-10 mRNA, although IFN-{gamma} message was expressed at a high level (Fig. 7Go, lane 3). For purpose of comparison, Fig. 7Go also includes RT-PCR data obtained with an encephalitogenic T cell line obtained from a LEW rat that had been immunized with MBP73–86 + CFA. This cell line also expressed IFN-{gamma}, TNF-{alpha}, and IL-10 mRNA (Fig. 7Go, lane 2). Thus, the two encephalitogenic T cell lines express mRNA and secrete the proinflammatory Th1 cytokines, whereas LEW/IFA, the T cell line derived from the EAE-tolerant LEW rat, did not secrete these cytokines. The finding that IFN-{gamma} message was expressed by the LEW/IFA cell line suggests posttranslational regulation of the production of this cytokine. We did not detect TGF-ß or IL-4 mRNA in either the DA/IFA or LEW/IFA cell line (data not presented).



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  		FIGURE 7. RT-PCR. Expression of cytokine mRNA derived from T cell lines DA/IFA (lane 1) and LEW/IFA (lane 3) immunized with peptide in IFA, compared with a T cell line derived from an MBP73–86 + CFA-immunized LEW rat with EAE (lane 2). A, ß-actin; (B) IFN-{gamma}; (C) TNF-{alpha}; and (D) IL-10. IFN-{gamma}, ß-actin, and IL-10 were run for 35 cycles; TNF-{alpha} was run for 44 cycles.

 
Finally, we attempted to induce tolerance in DA rats with MBP63–81-coupled splenocytes. As shown in Table IIGo, all DA rats injected i.v. with 5 x 107 MBP63–81-ECDI-splenocytes developed paralytic EAE when subsequently challenged with MBP63–81 + CFA (CFA was employed to ensure maximal encephalitogenicity of the emulsion). In contrast, significant protection against EAE was observed in LEW rats tolerized by i.v. injection of MBP68–86-ECDI-splenocytes, but not "sham" splenocytes, when challenged with MBP68–86 + CFA. This is consistent with the findings of Malotky et al. (13) that MBP68–86-ECDI-splenocytes elicit tolerance to EAE in LEW rats.


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  		Table II. Injection of encephalitogenic peptide-coupled splenocytes (SpC) i.v. elicits tolerance in LEW but not DA rats

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding in the present study is that DA rats appear to be resistant to the induction of immunological tolerance using two well-characterized procedures for eliciting unresponsiveness. First, we observed that DA rats develop EAE following immunization with MBP63–81 when given in an emulsion with IFA, i.e., adjuvant without mycobacteria. This finding stands in stark contrast to the previous reports that MBP or MBP-derived peptide given in IFA induces a profound state of immunological tolerance in Lewis rats, in susceptible strains of mice, and in guinea pigs (4, 5, 6, 7). It is apparent that the administration of peptide in IFA to DA rats induces a Th1-like response, as reflected by vigorous T cell proliferation, secretion of Th1-associated cytokines (TNF-{alpha} and IFN-{gamma}), and the induction of EAE. In contrast, neither of these proinflammatory cytokines was secreted by a T cell line derived from LEW rats immunized with MBP peptide in IFA, although IFN-{gamma} mRNA was expressed. This suggests that peptide/IFA-primed LEW rats do not develop EAE because their T cells do not translate the IFN-{gamma} message, and that the mycobacterial component of CFA provides the signals necessary to complete this process and subsequently activate LEW T cells to become encephalitogenic.

The T cell line derived from DA rats immunized with peptide in IFA also expressed IL-10 mRNA and secreted this cytokine. Although IL-10 is believed to function as a regulatory cytokine in murine models, it has previously been detected in encephalitogenic LEW rat T cell lines, and appears to depend on the nature of the antigenic stimulus (26). This suggests that IL-10 may not be an immunoregulatory cytokine for rats.

Our early work in the LEW rat (6), and that of Bernard in murine EAE (7), suggested that suppressor T cells mediate protection against EAE, because tolerance could be transferred to naive recipients with T cells from Ag-IFA-treated donors.

This subject has recently been reinvestigated by Forsthuber et al., who demonstrated that tolerization with MBP in IFA induces immune deviation from a Th1 to a Th2 response (8). Thus, protection conferred by "suppressor" T cells may be mediated by peptide-IFA-induced Th2 cells that secrete cytokines (e.g., IL-4, IL-5, and IL-10), which down-regulate the inflammatory Th1 cells that are responsible for autoimmune pathology. Furthermore, it has recently been reported that a Th2 response could be elicited by preimmunization with an unrelated Ag in IFA (27). Thus, mice primed with keyhole limpet hemocyanin (KLH) in IFA developed KLH-specific memory Th2 cells that secreted IL-4. These Th2 cells suppressed EAE provided that KLH was included in the encephalitogenic inoculum employed to challenge the mice. IL-4-secreting Th2 cells have also been implicated in tolerance to EAE induced by feeding myelin (28). However, other mechanisms may also be operative, because tolerance can be achieved in IL-4 deficient mice (10). The tolerized T cells regain encephalitogenic activity upon transfer to tolerogen-free recipients, suggesting that the peptide may render the encephalitogenic T cells anergic in the tolerized hosts.

The finding that encephalitogenic Ag + IFA elicits EAE in DA rats challenges the paradigm that this immunization protocol is inevitably tolerogenic, or necessarily results in preferential induction of Th2 responses (9), suggesting instead that the genetics of the host may override the effects of adjuvant relative to the induction of autoreactive T cell responses. Although both strains are susceptible to EAE, LEW and DA rats differ in MHC and background genes (1). DA rats are not rendered tolerant by peptide + IFA or peptide-ECDI-splenocytes (this report). They are also exquisitely susceptible to collagen-induced arthritis (29). Moreover, it has been reported that nasal tolerance can be induced in DA rats with MBP before challenge with an encephalitogenic emulsion, whereas nasal administration after onset of EAE exacerbates the disease (30). These findings suggest that DA rats may possess unique immunological and/or genetic characteristics that facilitate the induction of autoimmune diseases. Nevertheless, autoimmune diseases do not develop spontaneously in DA rats, indicating that self tolerance is tightly regulated. Recent evidence from our laboratory suggests that NK cells may be involved in the maintenance of immunological homeostasis in DA rats (31).

We were unable to induce tolerance to MBP63–81 in DA rats given MBP63–81-ECDI-splenocytes i.v., whereas, in confirmation of previous findings (13), LEW rats were protected against EAE when similarly treated with MBP68–86-ECDI-splenocytes (Table IIGo). The failure to induce unresponsiveness using two well-established protocols suggests that DA rats exhibit a generalized resistance to tolerance induction, although it has been reported that they can be tolerized by nasal instillation of MBP (30). Perhaps the susceptibility of DA, but not LEW rats to EAE following immunization with encephalitogenic peptide + IFA may reflect strain differences in APCs. Ridge et al. (32) reported that T cells can be primed by "professional" APCs (e.g., dendritic cells) whereas they are tolerized by noncostimulatory cells. However, our findings suggest that the unique susceptibility of DA rats to EAE cannot be explained on the basis of less stringent CD28/B7 costimulatory requirements (Figs. 4Go and 5Go). It is also possible that self reactive DA and LEW rat T cells are initially anergic, but that the state of anergy of DA T cells is more readily reversed by self Ag (33). One might speculate that the TCRs of the DA subset recognizing MBP63–81 may possess a greater overall avidity for peptide/MHC class II complexes than the LEW subset that recognizes MBP73–86. Stronger TCR avidity would allow for more stable interactions with APCs, obviating the need for increased immunogenicity of the immunizing emulsion afforded by the presence of mycobacteria.

The present findings may also be relevant to human diseases. For example, it is conceivable that diseases such as multiple sclerosis might develop more readily in a subgroup of individuals in whom tolerance is difficult to induce. Thus, it would seem worthwhile to ascertain whether patients with MS display genetically determined tolerance defects.

Furthermore, the induction of EAE without the requirement for mycobacteria should also facilitate studies of early T cell activation events that lead to EAE and avoid unknown and possibly artifactual effects attributable to mycobacterial products present in adjuvant. These studies are currently in progress.


   Acknowledgments
 
We thank Kathy Baran and Linda Walowicz of the Division of Laboratory Animal Resources for expert technical assistance.


   Footnotes
 
1 This study was supported by research Grant NS06985-32, from the National Institutes of Health, and Grant 1073-G10, from the National Multiple Sclerosis Society. R.H.S. is the recipient of a Javits Neuroscience Investigator Award from the National Institute of Neurological Diseases and Stroke. Back

2 Address correspondence and reprint requests to Dr. Robert H. Swanborg, Department of Immunology and Microbiology, Wayne State Universtiy School of Medicine, 540 East Canfield, Room 7263, Detroit, MI 48201. Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; ECDI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl; SI, stimulation index; KLH, keyhole limpet hemocyanin. Back

Received for publication April 15, 1999. Accepted for publication May 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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