An early expansion of CD8αβ T cells, but depletion of resident CD8αα T cells, occurs in the intestinal epithelium during primary simian immunodeficiency virus infection
Mattapallil, Joseph John; Reay, Elizabeth; Dandekar, Satya
From the Department of Internal Medicine, Division of Infectious Diseases, School of Medicine, University of California, Davis, CA, USA.
Sponsorship: Supported by grants from the National Institutes of Health (R01-DK43183, R01-AI43274 and RR-00169) and the Universitywide AIDS Research Program (F98-D-097), University of California.
Requests for reprints to: S. Dandekar, Division of Infectious Diseases, Room 3143, Tupper Hall, School of Medicine, University of California Davis, Davis, CA 95616, USA.
Received: 28 September 1999;
revised: 17 January 2000; accepted: 19 January 2000.
Objectives: To evaluate changes in the phenotypic heterogeneity and function of CD8 T cells in the intestinal epithelium during primary SIV infection.
Design: Previous studies have shown an increased prevalence of CD8 T cells in the intestinal epithelium in HIV and SIV infections. As intestinal CD8 T cells are a heterogeneous population we evaluated their phenotypic distribution (CD8αβ, CD8αα) and function [interferon (IFN)-γ production] during primary SIV infection.
Methods: The phenotype and functional potential of CD8 intestinal intraepithelial lymphocytes (IEL) prior to and following SIV infection were determined using flow cytometry.
Results: IEL were found to harbor CD8αβCD3, CD8ααCD3 and CD8αα+CD3− T-cell subsets. Most of the CD8CD4 double positive IEL expressed CD8αα homodimers. In primary SIV infection the frequency of CD8αβCD3 T cells increased dramatically whereas the frequency of CD8αα T cells declined. A higher frequency of CD8αβKi-67 IEL was observed following SIV infection suggesting that local cell proliferation might have contributed to an increased prevalence of CD8αβ IEL. In contrast, a severe depletion of CD8ααCD4 IEL occurred which contributed to the depletion of CD8αα IEL. The CD8αβ IEL were the major producers of IFN-γ in the intestinal epithelium and the frequency of IFN-γ-producing CD8αβ IEL was enhanced considerably in primary infection.
Conclusions: CD8αβ IEL may be important in generating early antiviral responses at the intestinal epithelium. However, alterations in CD8 T-cell subsets and their function may reflect early immunopathogenic events in the intestinal mucosa.
CD8 T cells are central to the development of anti-retroviral immune responses. Numerous studies have demonstrated that CD8 cytotoxic T lymphocyte (CTL) responses play an important role in controlling viral replication during primary HIV and SIV infections [1–5]. CD8 T cells are also the primary producers of cytokines such as interferon (IFN)-γ and MIP-1β during SIV/HIV infection [6,7], and secrete suppressive factors that may control viral replication [1,4,7]. These studies have demonstrated the importance of CD8 T cells in HIV and SIV infections.
The CD8 antigen is a dimeric molecule expressed as either CD8αα homodimers or CD8αβ heterodimers. Intestinal epithelium of humans [8,9] and mice [10–12] has been shown to harbor CD8αα and CD8αβ subsets of intraepithelial lymphocytes (IEL). Using athymic and nude mice, studies have shown that the CD8αα T cells in intestinal epithelium were thymus independent whereas the CD8αβ T cells were thymus dependent [10–13]. Studies have shown that the CD8αβ IEL were mature T cells capable of CTL activity [14–16] and cytokine expression . On the other hand, studies have suggested that CD8αα IEL were immature extrathymic T cells at various stages of development [11,18,19]. In contrast, other studies have reported that CD8αα IEL were mature CD8 T cells capable of both CTL activity [14,16] and cytokine production . These various subsets of CD8 T cells may interact to maintain homeostasis in intestinal mucosa and play an important role in intestinal mucosa-associated immune defense. The effect of HIV infection on the phenotype and functional potential of the various CD8 T-cell subsets in intestinal mucosa have not been evaluated.
HIV and SIV infections are characterized by a generalized CD8 lymphocytosis in both blood and mucosal lymphoid tissues [20–23]. Using the rhesus macaque model for HIV infection, we [6,24] and others  have demonstrated a similar increase in the prevalence of CD8 T cells in intestinal mucosa during primary SIV infection. However, information is limited regarding the infection-associated changes in the CD8 T-cell subsets contributing to the CD8 lymphocytosis.
The objective of this study was to evaluate the phenotypic and functional changes in CD8 IEL subsets following SIV infection of rhesus macaques. The expression of CD8α, CD8β, CD3, CD4, Ki-67 and IFN-γ production by intestinal IEL was determined by flow cytometry. Our results demonstrated that primary SIV infection led to a decline in the frequency of resident CD8αα IEL whereas the frequency of CD8αβ IEL increased dramatically. A higher proportion of the CD8αβ T cells expressed the Ki-67 antigen whereas a severe depletion of CD8ααCD4 T cells was observed during primary infection. The CD8αβ T cells were the primary producers of IFN-γ indicating that these IEL may play a role in the immunopathogenesis of SIV infection in intestinal mucosa.
Materials and methods
Animals, virus and tissue collection
Colony-bred rhesus macaques (Macaca mulatta) from the California Regional Primate Research Center (Davis, California, USA) were used in this study. The animals were housed in accordance with American Association for Accreditation of Laboratory Animal Care guidelines. Animals were seronegative for simian retrovirus and simian T cell leukemia virus type-1. Ten animals were infected with 10–100 animal infectious doses of uncloned pathogenic SIVmac251. Jejunum biopsy samples were obtained from six uninfected animals (n = 6). Longitudinal biopsy samples were obtained from two SIV-infected animals at 1 (n = 2) and 2 (n = 2) weeks post-infection (p.i.). In addition, jejunum tissue samples were collected at the necropsy time point from eight SIV-infected animals at 1 (n = 2), 2 (n = 2) and 8 weeks (n = 4) p.i. Tissue samples were immediately frozen for determination of viral loads. Samples were formalin fixed and also embedded in OCT for immunohistochemical analysis.
Quantification of plasma and tissue SIV RNA by branched DNA (bDNA) signal amplification assay
The bDNA assay specific for SIV was used to determine SIV RNA copy number in plasma and tissue samples as described previously [26–29]. Briefly, a standard curve for the SIV RNA copies was generated using serial dilutions of SIV-infected tissue culture supernatant containing cell-free SIV. The quantification for this standard curve was obtained by comparison with purified, quantified in vitro-transcribed SIVmac239pol RNA. Copy numbers of SIV RNA associated with viral particles in plasma samples were determined by comparison with the standard curve and reported as SIV RNA copies/ml plasma. Jejunal tissue samples were homogenized in guanidine hydrochloride buffer and SIV RNA copy numbers were quantified [27–29]. The SIV RNA burden in tissues was reported as SIV RNA copies/10 mg tissue. The lowest limit of quantification was around 1500 copies/ml of plasma and around 3000 copies/10 mg tissue.
Immunohistochemistry for CD8, CD3 and Ki-67 antigens
The CD8 T cells were localized in OCT embedded jejunal tissue sections using mouse anti-human CD8 (Dako Corporation, Carpentieria, California, USA) monoclonal antibody (mAb). Immunohistochemistry was performed with labeled streptavidin biotin (LSAB Kit; Dako Corporation) using the streptavidin–biotin–peroxidase complex technique with 3,3′-diaminobenzidine as chromogen. Endogenous peroxidase was inactivated with 3% hydrogen peroxide for 20 min. Control samples included the use of isotype control mAb, omission of primary antibodies and use of jejunal tissue sections from uninfected control animals.
Formalin fixed paraffin embedded tissue sections were used to localize CD3Ki-67 T cells in jejunal mucosa using a double staining technique. Jejunal tissue sections were immunostained for CD3 (Dako Corporation) as described previously [30,31]. Immunohistochemistry was performed with the LSAB Kit using the avidin–biotin–alkaline phosphatase complex assay and nitro blue tetrazolium/5-bromo-4-chloroindol-2-yl phosphate as chromogen (Boehringer Mannheim, Indianapolis, Indiana, USA). After immunostaining for CD3, Ki-67 cells were localized using anti-human Ki-67 mAb (MIB-1, Coulter Immunotech, Miami, Florida, USA) as described above for CD8 antigen. Control samples included use of isotype control mAb, omission of primary antibodies and use of jejunal tissue sections from uninfected control animals.
Isolation of IEL
IEL were isolated according to previously published procedures . Jejunum tissue samples were placed into IEL isolation medium (1 × Hank's balanced salt solution; Gibco, Grand Island, New York, USA) supplemented with 0.75 mM EDTA (Sigma Chemical Co., St. Louis, Missouri, USA), 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco), and 5% fetal calf serum (Gibco) at pH 7.2 and subjected to rapid shaking at 37°C for 30 min. Liberated cells were enriched for mononuclear cells using a 35 : 60 (v : v) isotonic discontinuous Percoll (Sigma Chemical Co.) density gradient. Mononuclear cells were found to band at the interface between the 35% and 60% fractions. More than 98% of the isolated mononuclear cells were viable as determined by trypan blue exclusion assay. The majority (88–94%) of the gated IEL were found to express αEβ7 antigen as recognized by the HML-1 antibody (Coulter Immunotech).
Monoclonal antibodies to human CD3 (Pharmingen, San Diego, California, USA), CD4 (Ortho-Diagnostics Systems Inc., Raritan, New Jersey, USA), CD8α (clone 3B5, Caltag Laboratories, South San Francisco, California, USA), CD8β (clone 2ST8.5H7; recognizes an epitope of CD8αβ heterodimer, Coulter Immunotech), Ki-67 (MIB-1 clone, Coulter Immunotech), IFN-γ (Pharmingen) were used in this study. Isotype controls were obtained from Caltag laboratories and Pharmingen.
Flow cytometric analysis of intestinal IEL
Freshly isolated cells from jejunum were stained with fluorescein isothiocyanate (FITC) conjugated anti-CD3 or anti-CD4, tricolor (TC)-conjugated anti-CD8α, and phycoerythrin (PE)-conjugated anti-CD8β according to methods described previously [6,28,29]. To determine the intracellular expression of Ki-67, unstimulated cells were labeled with anti-CD8α-TC and anti-CD8β-PE and fixed as described below. These fixed cells were permeabilized and labeled intracellularly with FITC-conjugated anti-Ki-67. Negative control samples included cells stained with matched isotype control antibodies.
Intracellular IFN-γ production was detected using IEL that were stimulated with 10 ng/ml phorbol myristate acetate (Sigma Chemical Co.) and 500 ng/ml ionomycin (Calbiochem, La Jolla, California, USA) in the presence of Monensin (2 μM) for 4 h [6,28]. After incubation, the cells were harvested and washed in cytoflow buffer (phosphate-buffered saline containing 1% bovine serum albumin). Cells were labeled with anti-CD8α-TC and anti-CD8β-PE and incubated for 30 min at 4°C. After washing in phosphate-buffered saline, cells were fixed (Cell Perm & Fix Kit, Caltag Laboratories), permeabilized and labeled with anti-IFNγ-FITC (Cell Perm & Fix Kit). Negative controls included samples stained with isotype control mAb. Cells were also fixed and labeled with anti-human IFN-γ or isotype control mAb without permeabilizing to ensure that only intracellular proteins were being labeled.
Cells prepared for both immunophenotypic analysis and intracellular proteins were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountainview, California, USA). Two thousand to 4000 events were collected in list mode after simultaneously gating on lymphocytes based upon their forward and light scatter characteristics and FL3 (CD8α). Collected data were analyzed using the Cell Quest software (Becton Dickinson).
Jejunal mucosa of SIV-infected animals had high viral loads
SIV RNA copy numbers were determined in plasma and jejunal tissue samples at 1, 2 and 8 weeks p.i. using the bDNA assay (Fig. 1). High viral loads were detected in plasma at 1, 2 and 8 weeks p.i., whereas high SIV RNA copy numbers were found in jejunal tissue at 2 and 8 weeks p.i.
A severe depletion of CD4 T cells in intestinal mucosa was accompanied by CD8 lymphocytosis in primary SIV infection
The frequencies of CD4 and CD8 T cells in jejunal epithelium were determined following SIV infection and compared with those in uninfected animals. The jejunal IEL were found to comprise 8–17% CD4 single positive T cells, whereas the frequency of CD4CD8 T cells ranged from 5 to 13% (data not shown). Following SIV infection, a severe depletion of CD4 and CD4CD8 T cells was observed (0–5%) in the intestinal epithelium as early as 2 weeks p.i. (data not shown). The depletion of CD4 and CD4CD8 T cells was accompanied by an increase in the prevalence of CD8 T cells at 2 and 8 weeks p.i. Immunohistochemical analysis revealed the presence of an increased number of CD8 T cells in the intestinal epithelium of SIV-infected animals at 2 (Fig. 2) and 8 weeks p.i. as compared with uninfected animals.
Depletion of CD8αα T cells in intestinal epithelium was accompanied by an increase of CD8αβ T cells during primary SIV infection
To determine the nature of SIV-associated CD8 T cell lymphocytosis in intestinal epithelium, the phenotype of CD8 T-cell subsets was determined using flow cytometry and compared with those of uninfected controls. Two major subsets of CD8 T cells were observed on the basis of the CD8α and CD8β chain expression. One subset of CD8 T cells was found to express both the CD8α and β chains (CD8αβ), whereas other subset of CD8 T cells expressed only CD8α chain (CD8α+β− ). As CD8 IEL exist as either CD8αα homodimers or CD8αβ heterodimers, this indicates that the CD8α+β− subset of IEL were CD8αα T cells. In uninfected control animals, 66 ± 6% of CD8 IEL were CD8αβ T cells, whereas 35 ± 11% were CD8αα T cells (Table 1). All of the CD8αβ T cells co-expressed CD3 whereas the CD8αα T cells harbored CD3 and CD3− subsets of CD8 T cells with a majority of them being CD3.
Distribution of CD8 T-cell subsets of IEL was altered following SIV infection (Table 1). As compared with uninfected controls, frequency of CD8αβ T cells increased dramatically at 2 (88 ± 7%) and 8 weeks p.i. (93 ± 4%). In contrast, the frequency of CD8αα T cells declined at 2 (14 ± 3%) and 8 weeks p.i. (9 ± 4%). The majority of this increase in CD8 IEL was attributable to an expansion of the CD8αβCD3 subset (Table 1). In contrast, both the CD8ααCD3 and CD8αα+CD3– subsets declined as early as 2 weeks p.i.
Depletion of CD8αα IEL of SIV-infected animals correlated with decline in CD8CD4 double positive T cells
To determine whether the depletion of CD8αα in SIV infection could be attributed to the depletion of CD4CD8 double positive T cells, the expression of CD4 on CD8αα and CD8αβ T cells was examined (Table 1). Flow cytometric analysis revealed that most of the CD8CD4 T cells were CD8αα. A severe depletion of CD8ααCD4 T cells was observed at 2 and 8 weeks p.i. A representative dot plot is shown in Fig. 3a.
Local proliferation of CD8αβ+ IEL occurred in primary SIV infection
To determine whether local proliferation contributed to an increase in CD8αβ T cells in the intestinal epithelium, expression of Ki-67 was examined in CD8αα and CD8αβ T cells prior to and following SIV infection (Table 1). In uninfected animals, a minor proportion of CD8αα and CD8αβ T-cell subsets was found to express the Ki-67 antigen. However, the frequency of CD8αβKi-67 cells increased as early as 2 weeks following SIV infection (Table 1). No detectable change was noted in the frequency of CD8ααKi-67 cells. A representative dot plot is shown in Fig. 3b. Immunohistochemical analysis revealed the presence of a higher number of CD3Ki-67 cells in intestinal epithelium of SIV-infected animals at 2 (Fig. 2) and 8 weeks p.i. As most of the CD4 T cells were depleted at 2 and 8 weeks p.i., this would suggest that the majority of CD3Ki-67 cells were CD8 T cells.
A higher frequency of IFN-γ producing CD8αβ IEL was found in primary SIV infection
To determine the frequency and phenotype of IFN-γ-producing CD8 T cell subsets in intestinal epithelium, we examined the potential of CD8αα and CD8αβ T-cell subsets to produce IFN-γ (Table 1). Our results demonstrated that a higher proportion of CD8αβ T cells in uninfected animals was primed to produce IFN-γ as compared with CD8αα T cells. Following SIV infection, the frequency of IFN-γ-producing CD8αβ T cells was found to increase at 2 weeks p.i. and remained high at 8 weeks p.i. In contrast no change was observed in the potential of CD8αα T cells to produce IFN-γ. A representative dot plot is shown in Fig. 4.
To determine the phenotype of the IFN-γ-producing subset of CD8 T cells, analysis gates were set to include only IFN-γ-positive lymphocytes. Our results demonstrated that 41–46% of IFN-γ-producing lymphocytes in uninfected animals were CD8αβ T cells whereas 8–11% were CD8αα T cells. Following SIV infection 42–72% of IFN-γ-producing lymphocytes were CD8αβ T cells at 1 week p.i., whereas 75–88% and 72–87% of IFN-γ-producing lymphocytes were CD8αβ T cells at 2 and 8 weeks p.i. In contrast, CD8αα T cells were minor producers of IFN-γ at 1 (12–27%), 2 (8–13%) and 8 weeks p.i. (6–10%).
In primary SIV infection, high SIV RNA loads were accompanied by a severe depletion of CD4 T cells in intestinal epithelium. A coincident increase in the prevalence of CD8 T cells was observed as reported previously [6,24]. The CD8 T cells in the intestinal epithelium of rhesus macaques were found to be of a heterogeneous phenotype. Two major subsets of CD8 T cells were detected: CD8αα and CD8αβ; a higher proportion was CD8αβ T cells. All of the CD8αβ T cells were found to co-express CD3, whereas both CD3 and CD3– subsets of CD8αα T cells were observed (Table 1). Previous studies in mice have shown that approximately 70% of CD8 IEL were CD8αβ and the remaining 30% were CD8αα T cells . The human intestinal epithelium has been shown to harbor 5–37% CD8αα T cells , whereas approximately 67% of CD8 IEL were CD8αβ T cells . These subsets of CD8 T cells may contribute to maintaining homeostasis in the intestinal epithelium.
Following SIV infection, a dramatic increase was observed in the frequency of CD8αβCD3 T cells as early as 2 weeks p.i. whereas the proportions of CD8αα T cells declined (Table 1). High viral loads in the intestine during primary SIV infection may contribute towards an increased prevalence of CD8αβCD3 T cells in intestinal epithelium. Previous studies have shown that peripheral blood CD8αβ T cells harbored SIV-specific CTL activity . Our studies had demonstrated that primary intestinal IEL displayed strong SIV-specific CTL activity as early as 2 weeks p.i. . The increased prevalence of the CD8αβ T cells in response to viral infection could contribute to CTL responses in the intestinal mucosa. Our results demonstrated that a higher frequency of CD8αβ T cells expressed the Ki-67 antigen at 2 and 8 weeks p.i. (Fig. 3b) suggesting that local proliferation may be a mechanism for the increased prevalence of CD8αβ T cells within intestinal epithelium. Poussier et al. reported that the CD8αβ T cells in intestinal epithelium of mice had a higher proliferative capacity to antigenic stimuli than the CD8αα T cells. Thus, although CD8αα T cells may harbor subsets of activated T cells, the CD8αα T cells may be terminally differentiated T cells incapable of further proliferation. The role of trafficking in this process however cannot be ruled out. Although analysis of peripheral blood was not undertaken in the present study, previous studies have reported that peripheral blood of humans  and rhesus macaques  harbors CD8αβ T cells. These subsets of CD8 T cells could potentially migrate into intestinal mucosa following SIV infection. The CD8αβ IEL could also be generated from the resident progenitor cells located in the intestinal epithelium. The intestinal epithelium of rhesus macaques has been shown to harbor a subset of CD34 progenitor cells . The frequency of CD8 committed CD34 progenitor cells was found to increase during early SIV infection suggesting that this may be a mechanism for the increased prevalence of CD8αβ T cells in the intestinal epithelium.
Interestingly, primary SIV infection led to the depletion of CD8αα T cells. In order to determine the mechanism of CD8αα T cell depletion, we examined the expression of CD4 on the CD8αα and CD8αβ T cells. Our results demonstrated that most of the CD8CD4 double positive T cells in the intestinal epithelium of rhesus macaques were CD8αα T cells (Table 1). Previous studies in mice  and humans  have shown that the CD8CD4 double positive T cells in intestinal mucosa were primarily CD8αα T cells. A severe depletion of CD8ααCD4 T cells was observed as early as 2 weeks p.i. (Fig. 3a) suggesting that this may be a mechanism for the depletion of CD8αα IEL. Direct viral cytotoxicity may be playing a role in this process. However, it is possible that activation-induced cell death or apoptosis may contribute to the depletion of this subset of IEL. Some CD8αα cells could also migrate to the periphery during primary SIV infection. Studies using the feline model have shown that the proportions of CD8αα T cells (CD8α+β–) increased in peripheral blood during chronic infection . The significance of the depletion of CD8αα T cells in SIV infection is difficult to determine; however, studies in mice have suggested that CD8αα IEL were immature extrathymic T-cell subsets that may differentiate through a CD4CD8αα stage into mature CD4 and CD8αβ T cells in intestinal epithelium . Rozing and De Geus  have suggested that the CD4CD8αα cells found in mice were intermediates in the extrathymic differentiation pathway for the local generation of resident CD8αα IEL. The depletion of CD8ααCD4 T-cell subsets during primary SIV infection would suggest that the extrathymic development pathway for regeneration of resident CD8 IEL subsets was being compromised. On the other hand, studies have suggested that CD8αα IEL were mature T-cell subsets. Fujihashi et al. had shown that the murine CD4CD8 IEL expressed a Th2 type of cytokine profile. As CD4CD8 IEL are primarily CD8αα, a TH2 type of cytokine profile would suggest an anti-inflammatory role for this subset of IEL. The above studies indicate that CD8αα intestinal IEL are a unique subset of T cells specific to the intestinal epithelium and that develop in intestinal microenvironment; hence, they may have unique immune regulatory functions at this site. The depletion of this subset of T cells could have an adverse effect on T-cell homeostasis in intestinal epithelium thereby contributing to the immunopathogenesis of HIV/SIV infections.
We have previously reported that CD8 IEL were the primary producers of IFN-γ and that their potential to produce IFN-γ was up-regulated during primary SIV infection . Limited information is available regarding the exact phenotype of IFN-γ-producing CD8 IEL. Our results demonstrated that CD8αβ IEL were the primary producers of IFN-γ. Further, the frequency of IFN-γ-producing CD8αβ T cells increased as early as 2 weeks p.i. (Fig. 4) suggesting that CD8αβ IEL may play an important role in generating antiviral cytokine responses during primary SIV infection. Studies in mice have shown that CD8αβ IEL secreted higher levels of cytokines such as IFN-γ and tumor necrosis factor-α[17,39] as compared with CD8αα IEL. Our results demonstrated that only a minor subset of the resident CD8αα T cells were capable of producing IFN-γ as compared with the CD8αβ IEL. Following SIV infection no major changes were observed in the frequency of IFN-γ-producing CD8αα IEL. Studies in mice [18,19,40] have suggested that the CD8αα IEL were non-responsive to antigenic or anti-T-cell receptor stimulation.
In conclusion, primary SIV infection was found to alter the phenotypic profile of CD8 T cell subsets in intestinal epithelium leading to an increased prevalence of CD8αβ T cells and a decline in CD8αα T cells. Local proliferation of CD8αβ T cells and the severe depletion of the CD8ααCD4 T cells may partially account for altered T-cell homeostasis in the intestinal epithelium following SIV infection. Further, our results showed that the potential of CD8αβ IEL to produce IFN-γ was up-regulated during primary SIV infection. Taken together, these results suggest that primary SIV infection led to an increase in the prevalence of CD8αβ IEL that were capable of producing IFN-γ. The IFN-γ produced by these T cells, though protective may potentially contribute to the immunopathogenesis of SIV and HIV infections. IFN-γ is a proinflammatory cytokine that has been shown to play a central role in the development of cellular immune responses leading to the elimination of intracellular pathogens [41–43]. The IFN-γ has been shown to mediate anti-viral immunity by lysis of infected cells by CTL activity [44,45]. On the other hand, an increased prevalence of IFN-γ producing CD8αβ IEL may indicate abnormal production that may directly alter the integrity of the intestinal epithelium by increasing the permeability of epithelial cell–cell tight junctions . Further, IFN-γ has been reported to have a potentiating effect on macrophages and neutrophils, and stimulate the synthesis and release of reactive oxygen metabolites [47,48] that may lead to the development of inflammatory conditions in the intestinal mucosa.
The authors thank L. Hirst, R. Tarara, D. Canfield and J. Bernal at the University of California Davis, and P. Dailey at Chiron Corporation for their valuable assistance in the project.
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