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. 1999 Feb;67(2):618–623. doi: 10.1128/iai.67.2.618-623.1999

A Controlled Clinical Study of the Effect of Nasal Immunization with a Streptococcus mutans Antigen Alone or Incorporated into Liposomes on Induction of Immune Responses

Noel K Childers 1,*, Giang Tong 1, Stephen Mitchell 1, Katharine Kirk 2, Michael W Russell 3, Suzanne M Michalek 3
Editor: V A Fischetti
PMCID: PMC96363  PMID: 9916067

Abstract

Recent attention to mucosal immunization strategies has been focused on the nasal route for vaccine delivery. This study was designed to determine the effectiveness of a liposome-protein vaccine compared to that of a protein-only vaccine in inducing immune responses in humans. Healthy subjects were randomly assigned to two groups and immunized intranasally with a crude antigen preparation rich in glucosyltransferase (C-GTF) from Streptococcus mutans, alone or in liposomes. Parotid saliva, nasal wash, and serum were collected prior to and at weekly intervals following immunization and were analyzed for anti-C-GTF activity by enzyme-linked immunosorbent assay. The levels of immunoglobulin A (IgA) anti-C-GTF activity in the nasal wash from both groups after immunization increased to a mean peak of fivefold over the baseline level on day 28. Salivary IgA anti-C-GTF responses were induced to a lesser extent. IgG and IgA anti-C-GTF responses in serum were detected on day 14. The IgA responses were predominantly of the IgA1 subclass. These results show that C-GTF vaccines were more effective in inducing a local secretory IgA antibody response than a salivary or serum response when they were given intranasally. The IgA1 anti-C-GTF response in nasal wash samples for liposomal antigen versus antigen only was the only response which was significantly different (P < 0.04). This suggests that the form of the antigen affects the magnitude of the local mucosal response but not that of a disseminated response. These results provide evidence for the effective use of a nasal protein vaccine in humans for the induction of mucosal and systemic responses.

Dental caries is considered to be one of the most prevalent and costly infectious diseases in the world (16), and despite the reports that this disease is on the decline in some developed countries (34, 47), it continues to be a worldwide problem. Secretory immunoglobulin A (IgA) antibodies in saliva are considered the first line of defense against pathogens present in the oral cavity, including Streptococcus mutans, the principal etiologic agent of dental caries. Therefore, studies aimed at the development of a caries vaccine have focused on the use of immunization regimens which stimulate the induction of IgA responses in saliva.

Oral administration of a vaccine results in the induction of IgA responses in various secretions, including saliva, via the common mucosal immune system (7). Studies in humans have shown that oral immunization with antigens from S. mutans results in mucosal IgA responses (8, 11, 43); however, the magnitude of the immune responses was shown to be low and their persistence limited. Recently, interest has been directed toward determining the importance of nasal immunization as a route for inducing mucosal responses, especially in the upper respiratory tract and oral cavity. The human nasal mucosa contains an abundance of IgA-secreting plasma cells (predominantly IgA1) which may originate in bronchus-associated lymphoid tissue or tonsils (5). The human nasal mucosa also contains T lymphocytes (48) and HLA-DR-expressing dendritic and epithelial cells (42), a fact which provides evidence that it may be an inductive site for responses in nasal (and oral) secretions. In this regard, intranasal (i.n.) immunization against respiratory pathogens (e.g., influenza and parainfluenza viruses) in humans results in antibody responses in nasal secretions and serum (12, 32). We have shown that i.n. immunization of humans with a crude preparation of an S. mutans antigen preparation rich in glucosyltransferase (C-GTF) in liposomes resulted in anti-C-GTF responses in nasal wash and saliva (9).

The present study compared the effectiveness of a liposomal preparation of S. mutans antigen to that of the free antigen in inducing mucosal and systemic immune responses after i.n. immunization of humans in a double-blind study.

MATERIALS AND METHODS

Bacteria, media, and reagents.

The C-GTF preparation used as the antigen in this study was derived from S. mutans GS-5 (a serotype c isolate, obtained from F. Macrina, Virginia Commonwealth University, Richmond, Va.) and was grown in streptococcal defined medium (J.R.H. Biosciences, Lenexa, Kans.) (45).

The components used for production of liposomes consisted of d,l-α-dipalmitoyl phosphatidylcholine, cholesterol, and dicetylphosphate (Sigma Chemical Company, St. Louis, Mo.). Liposomes were prepared by sonication of aqueous antigen suspensions and membrane filtration as previously reported (9).

Immunological reagents used for enzyme-linked immunosorbent assay (ELISA) analysis consisted of biotinylated goat anti-human IgA, IgM, and IgG (Biosource Inc., Burlingame, Calif.); unlabeled rabbit or goat anti-human IgA, IgM, and IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.); and mouse monoclonal IgG anti-human IgA1 and IgA2 (Accurate Chemical & Scientific Corp., Westbury, N.Y.). Biotinylated goat anti-mouse IgG (Southern Biotechnology Associates Inc., Birmingham, Ala.) was used as the detecting antibody for the IgA subclass analyses. Fetal calf serum (Flow Laboratories Inc., McLean, Va.) was used as the blocking reagent in the ELISA.

Antigen.

C-GTF used for immunization and ELISA was derived as previously reported (9). Briefly, following growth of S. mutans GS-5 in a 400-liter broth culture, cells were removed by centrifugation, the culture supernatant was concentrated by using a PLGC Pelicon cassette system (10,000 MW cutoff; Millipore Inc., Bedford, Mass.), and proteins were precipitated from the supernatant with 60% saturated ammonium sulfate. The purity, immunogenicity, and biologic activity of the resulting C-GTF preparation were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis with antisera to purified cloned glucan binding domain of GTF-I (24) and to purified antigen I/II (AgI/II) (raised against purified S. mutans AgI/II [IB162]) (39), and periodic acid-Schiff (PAS) staining following incubation with 10% sucrose, as previously reported (11). Purified AgI/II was derived from S. mutans IB162 as previously described (39).

Experimental design for human study.

Twenty-one healthy adult volunteers ranging in age from 20 to 45 years were recruited to participate in this study. In compliance with guidelines established by the University of Alabama at Birmingham (UAB) Institutional Review Board, informed consent was obtained from the subjects. All but three subjects (assigned numbers 5, 16, and 20) had previous experience with dental caries, although none had active or recent carious lesions prior to or during this study. One subject reported having had a tonsillectomy (subject 5), one had had an adenoidectomy (subject 18), and two had had a tonsillectomy and adenoidectomy (subjects 12 and 17) during childhood.

The 21 subjects were randomly assigned to group A (liposomal C-GTF; n = 11) or group B (free C-GTF; n = 10). The subjects were immunized i.n. twice (day 0 and day 7) with the designated preparation by having 68 μl deposited in each nostril (total dose of C-GTF, 250 μg) while the subject was reclined. Both the subject and the clinician were blind as to the group assignment. Unstimulated parotid saliva, nasal wash, and serum samples were collected weekly for 3 weeks before (baseline) and then weekly for 8 weeks following the initial immunization for analysis of anti-C-GTF antibody activity. Parotid saliva samples were obtained by using Schaeffer cups (41). Nasal wash was obtained by depositing 1.5 ml of sterile saline into each nostril while the subject was reclined and allowing it to remain in the nostril for approximately 10 s. The subject was then instructed to sit up, and the nasal wash solution was allowed to drain into a specimen cup. Saliva and nasal wash samples were immediately clarified by centrifugation at 14,000 × g in an Eppendorf centrifuge. Serum was obtained from centrifuged (14,000 × g) blood collected by finger stick in Microvette tubes with clotting activator (Sarstedt, Numbrecht, Germany). All samples were aliquoted and frozen at −70°C until used for ELISA.

Antibody analysis.

An ELISA was used to determine the levels of total immunoglobulin and the relative concentrations of antibodies to S. mutans C-GTF as previously described (11). Briefly, polyvinylchloride microtiter plates (Dynatech Laboratories Inc., Chantilly, Va.) were coated overnight at room temperature with goat or rabbit anti-human IgA, IgM, IgG, or C-GTF diluted in phosphate-buffered saline. Optimal dilutions of saliva, nasal wash, or serum (50 μl) in duplicates of four to eight twofold dilutions were added to designated wells of the microtiter plates. A human serum pool of known isotype concentrations (Dade Moni-Trol; Baxter Diagnostic Inc., Deerfield, Ill.) and purified human IgA1 or IgA2 myeloma protein (provided by J. Mestecky, UAB) were used as the total immunoglobulin standards. Following sample incubation, biotin-conjugated goat antiserum to human IgA, IgM, or IgG (Biosource) or mouse monoclonal anti-human IgA1 or IgA2 (Accurate) was added to the appropriate wells. Color development was accomplished with streptavidin-peroxidase conjugate (Southern Biotechnology Associates), followed by ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] substrate (Sigma) in citrate buffer and recorded at 414 nm. A four-parameter curve fitting program (Softmax; Molecular Devices) was used to construct reference curves for each ELISA plate from optical density readings of the standard plasma pool of known immunoglobulin concentration. Plasma results were reported in nanograms of anti-C-GTF antibody per ml, while saliva and nasal wash results were converted to a ratio of anti-C-GTF antibody per total isotype or subclass antibody concentration to normalize for variation in total immunoglobulin content in the samples. For each subject, results obtained from the three baseline samples were averaged for comparison to postimmunization anti-C-GTF responses (reported as a percent increase over baseline activity for each subject).

Statistics.

A mixed-model analysis was used for comparison of antibody activity. This analysis considers group (liposomal versus free antigen), time (e.g., preimmunization versus postimmunization), and group times time to be fixed effects and subject-to-subject variation and its interactions to be random effects. Due to the heterogeneity of variances and right-skewness for the original variables, their common logarithms were analyzed. Significant differences were determined to be P values less than 0.05.

RESULTS

All 21 individuals completed the study, and none reported any side effects during or after i.n. immunization.

Vaccine characterization.

S. mutans GS-5 grown in 400 liters of chemically defined medium yielded over 1 g of proteinaceous material. The relative purity and biological activity of the C-GTF preparation were confirmed by SDS-PAGE and PAS staining, respectively (Fig. 1). The Coomassie blue-stained gel revealed a broad major band at approximately 161 kDa for C-GTF and a 193-kDa band for AgI/II. PAS staining following 10% sucrose incubation resulted in a band corresponding with putative enzymatically active GTF at 161 kDa and smaller molecular mass moieties (approximately 109 and 86 kDa) also producing insoluble carbohydrate polymers as previously reported (10). No activity was seen with purified AgI/II.

FIG. 1.

FIG. 1

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SDS-PAGE of C-GTF and purified AgI/II from S. mutans separated on 7% resolving gel. Lanes 1 to 3 are a Coomassie blue staining of the molecular mass standard (lane 1), C-GTF (lane 2) and purified AgI/II (lane 3). Lanes 4 and 5 are a PAS staining of C-GTF (lane 4) and purified AgI/II (lane 5) after incubation with 10% sucrose. The numbers to the left, in kilodaltons, are molecular mass markers.

The C-GTF was further characterized by Western blot analysis with antiserum to purified AgI/II and to cloned glucan binding region of GTF-I (Fig. 2). Both antisera reacted with the C-GTF preparation, detecting bands in the molecular mass range of 150 to 165 kDa. The antisera to AgI/II but not those to GTF reacted with the AgI/II preparation. These results indicate the presence of GTF and a truncated AgI/II protein in the C-GTF preparation.

FIG. 2.

FIG. 2

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Western blot analysis of C-GTF (lanes 1 and 3) and purified AgI/II (lanes 2 and 4) with polyclonal antiserum raised against purified AgI/II (lanes 1 and 2) or cloned glucan binding domain of GTF-I (lanes 3 and 4). Lane 5 is the molecular mass standard. The numbers to the right, in kilodaltons, are molecular mass markers.

Nasal wash response.

A nasal IgA1 anti-C-GTF response was detected in 19 of 21 subjects (i.e., the response was at least twofold over baseline at some time points following immunization). Individuals given liposomes containing C-GTF (group A) had a higher response throughout the experiment than those given free C-GTF (group B) (Fig. 3). The difference in the response was significant (P < 0.04) on day 28.

FIG. 3.

FIG. 3

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Nasal IgA1 anti-C-GTF activity as a percentage of total IgA1 antibody activity before and after i.n. immunization. Values are the ratio of C-GTF-specific/total immunoglobulin for samples collected before and after i.n. immunization with liposomal C-GTF (group A [■]) or free C-GTF (group B [•]). ∗, mean difference between group A and group B values is significantly different (P < 0.04) on day 28.

Since no significant difference was seen between the two groups for all other analyses (time points, including serum and saliva), the data from groups A and B were combined. Preimmunization variability was found to be comparatively low when related to postimmunization data for the nasal wash IgA anti-C-GTF responses (Fig. 4a). Mixed-model analysis resulted in a highly significant (P < 0.0001) day effect, which corresponded to values obtained for the three preimmunization samples and that from day 7, in contrast to the values obtained with samples collected at the seven remaining time points (postresponse) with a mean peak of a fivefold increase in the ratio of IgA anti-C-GTF/total over baseline occurring on day 28.

FIG. 4.

FIG. 4

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(a) Nasal IgA anti-C-GTF activity as a percentage of total antibody activity before and after i.n. immunization. Values are the mean ratio of anti-C-GTF-specific/total immunoglobulin activity (plus standard deviation) in nasal wash samples collected from human subjects before and after i.n. immunization with C-GTF (combined data from liposomal C-GTF and free C-GTF groups). (b) Nasal IgA1 (□) and IgA2 (○) anti-C-GTF activity as a percentage of total antibody activity after i.n. immunization. Values are the mean percent increase in anti-C-GTF/total immunoglobulin activity in nasal wash samples (over three preimmunization samples) collected from human subjects after i.n. immunization with C-GTF (combined data from liposomal C-GTF and free C-GTF groups).

A highly significant (P < 0.0001) IgA subclass day (i.e., response) effect was also observed (Fig. 4b). The peak (day 28) percent increase in IgA1 compared to the IgA2 response was not only higher (fourfold versus twofold, respectively), but the magnitude of IgA1 (total and specific antibody activity; Table 1) was also ∼2 to 3 times that of IgA2.

TABLE 1.

Mucosal IgA subclass anti-C-GTF and total IgA levels before and after i.n. immunizationa

Sample Mean ± SD (range)
IgA1 anti-C-GTF (ng/ml) IgA2 anti-C-GTF (ng/ml) Total IgA1 (μg/ml) Total IgA2 (μg/ml)
Nasal wash
 Pre 50.8 ± 44.1 34.6 ± 42.2 39.8 ± 33.1 10.8 ± 8.1
 Post 137.7 ± 152.5 (0–708.8) 49.8 ± 49.1 (0–205.1) 36.0 ± 29.9 (5.3–240.5) 10.8 ± 11.8 (1.0–124.9)
Saliva
 Pre 436.6 ± 274.5 381.8 ± 304.9 272.2 ± 176.6 222.6 ± 227.2
 Post 611.3 ± 574.9 (0–5,247) 431.7 ± 390.6 (0–2,975) 278.5 ± 136.4 (16.3–2,000) 199.3 ± 156.1 (2.7–1,760)
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a

Values are levels of IgA subclass anti-C-GTF antibody and total IgA as determined by ELISA (see Materials and Methods) for samples collected before (Pre) and after (Post) immunization. 

Saliva response.

The salivary IgA anti-C-GTF activity in 9 of 21 subjects was at least twofold over baseline with a mean peak of a 73% IgA anti-C-GTF increase over baseline occurring on day 21. Although variable and relatively small, the change in the mean level of salivary IgA anti-C-GTF/total for four preresponse samples, compared to seven postresponse samples, was significantly different by mixed-model analysis (P < 0.0001; Fig. 5).

FIG. 5.

FIG. 5

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Salivary IgA anti-C-GTF activity as a percentage of total IgA antibody activity before and after i.n. immunization. Values are the mean ratio of C-GTF-specific/total immunoglobulin (plus standard deviation) in anti-C-GTF activity in parotid saliva samples collected from human subjects before and after i.n. immunization with C-GTF (combined data from liposomal C-GTF and free C-GTF groups).

The mean ratio of IgA1 anti-C-GTF/total response was a greater percent increase over baseline level than that of IgA2 (82 and 23%, respectively). Furthermore, the magnitudes of IgA1 and IgA2 antibody responses in the samples were similar, i.e., the ratio of IgA1/IgA2 was ≈1.2 to 1.4 (Table 1).

Serum response.

An IgA1 response in serum was detected in 13 of 21 subjects with a mean increase of 1.5- to 2-fold over baseline from day 21 to 56 following immunization (Fig. 6). IgA1 responses persisted through the termination of the experiment (day 56). No significant IgA2 antibody response was found in serum. The mean IgG response was of a lower magnitude (∼40% mean increase over baseline; Fig. 6), with only 4 of 21 subjects showing an increase of more than twofold over baseline anti-C-GTF activity.

FIG. 6.

FIG. 6

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Serum IgA1 (▴) and IgG (⧫) anti-C-GTF responses after i.n. immunization with liposomal C-GTF. Values are the percent increase in anti-C-GTF activity in serum samples (over three preimmunization samples) collected from human subjects after i.n. immunization with C-GTF (combined data from liposomal C-GTF and free C-GTF groups).

Responses to AgI/II.

An analysis by ELISA of saliva, serum, and nasal wash collected from individuals immunized with liposomal C-GTF preparation by using purified AgI/II as the coating antigen revealed response patterns similar to those obtained with the homologous antigen C-GTF (data not shown).

DISCUSSION

Historically, studies to promote the induction of salivary immune responses in humans by mucosal immunization have mainly concentrated on administering antigen via the gastric route (31). The rationale behind this approach was that oral immunization results in mucosal responses by way of the common mucosal immune system, i.e., antigen-sensitized lymphoid cells in the gut-associated lymphoid tissue (IgA inductive sites) are disseminated to various mucosal effector sites. Recent evidence, however, suggests that compartmentalization may exist in the mucosal immune system, whereby the response pattern seen at various mucosal effector sites may differ according to the route of mucosal immunization (30, 33). As a result of this information, much interest has been placed on determining the importance of Waldeyer’s ring as an inductive site for mucosal responses, especially for responses in the upper respiratory tract and oral cavity. Numerous reports on i.n. immunizations with antigens (19, 21, 22, 25, 29, 38, 40, 44, 49) or with liposomal antigens (1–3, 6, 14, 15, 20) involving various animal models have provided encouraging results, indicating that this type of immunization increases antigen-specific antibody responses in pulmonary and oral secretions.

The present study was initiated to investigate the effectiveness of immunizing humans via the i.n. route with S. mutans antigens, either incorporated into liposomes or free, in inducing salivary IgA responses. The present findings expanded our preliminary results (9) regarding the immunogenicity of C-GTF when given i.n. to five human subjects. The inclusion of a larger sample size greatly enhanced the statistical relevance of our findings. In this regard, we observed significantly higher mean postimmunization IgA anti-C-GTF activity in nasal wash, parotid saliva, and serum than that observed preimmunization.

This study also involved a blind clinical test of two forms of the vaccine antigen, i.e., liposomal and free antigen. Subjects given the liposomal C-GTF by the i.n. route had significantly higher nasal wash IgA1 immune response levels than volunteers given the free antigen. However, the finding that no significant difference in responses was detected in other samples suggests that soluble C-GTF antigen was as effective as liposomal C-GTF in inducing salivary and serum responses when given by the i.n. route. Alternatively, it is recognized that sample sizes are small in this study and that power may not have been sufficient to detect small differences.

Previous studies (9, 11) have shown that the culture supernatant of S. mutans GS-5 grown in chemically defined medium is enriched for GTF. An ammonium sulfate precipitation of this preparation results in a fraction which predominantly consists of a 165-kDa protein, determined by SDS-PAGE, is enzymatically active in the presence of sucrose, and is immunogenic when given to animals and humans. Some strains of S. mutans, including GS-5, are known to produce a truncated AgI/II surface protein (∼155 kDa) that is released into the culture medium (35). Biochemical and immunological analysis of the C-GTF antigen preparation indicate the presence of both GTF and truncated AgI/II. These analyses also indicated the presence of other antigens. The two additional bands indicating insoluble carbohydrates in the PAS stain of gels following incubation with sucrose may represent fructosyltransferase or glucan binding proteins. Furthermore, minor bands identified in Western blots may illustrate other antigens or breakdown products in the C-GTF preparation. Immunization of humans by the i.n. route with C-GTF resulted in the induction of immune responses to the immunogen and native AgI/II. Further studies are required to define the dynamics of the responses to purified GTF and AgI/II, as well as other antigens in the C-GTF which may have contributed to the immune responses detected, in different body fluids and the effectiveness of these responses in inhibiting S. mutans infection. Since GTF and AgI/II are considered virulence antigens of S. mutans, which are involved in two different stages of the pathogenesis of dental caries, i.e., sucrose-independent reversible attachment mediated by AgI/II and sucrose-dependent irreversible attachment mediated by GTF (36), the induction of immune responses to C-GTF could act against both stages. Therefore, the potential for an additive or synergistic protective benefit of the bivalency (or potentially multivalency) of the C-GTF used in this study is attractive in the continued development and evaluation of caries vaccines for use in humans.

An overall goal of our studies is to determine a mucosal route of immunization with an S. mutans vaccine that induces optimal, effective salivary immune responses. Although a significant increase was seen in the salivary IgA response after i.n. immunization, the response was lower than that seen in nasal wash and serum. It is possible that the salivary response induced would be effective in protecting against S. mutans infection, as suggested by the results of experimental animal studies (31). Nevertheless, further studies are needed to identify a mucosal immunization route, dosage, antigen form, adjuvant, and timing schedule for further enhancing the magnitude and longevity of the salivary response. These studies may involve additional testing of i.n. immunization strategies of other routes.

Since i.n. immunization resulted in primarily a nasal response, there may exist a mucosal IgA inductive site which preferentially promotes a salivary response. Fukuizumi and coworkers (17) have presented data that indicate that tonsils may play a role in the selective induction of oral responses. In that study, rabbits were immunized with sheep erythrocytes by the i.n. or tonsillar (pharyngeal) route. Antibody activity was seen in nasal washes after nasal immunization, while salivary responses resulted from tonsillar immunization (antigens topically applied via the oral cavity). In a recent study these investigators, using S. mutans whole cell antigen, also reported the induction of a salivary response following tonsillar immunization (18).

The tonsils are a potential induction site for responses within the oral cavity. Waldeyer’s ring (consisting of palatine, lingual, and nasopharyngeal [adenoids] tonsils), located at the proximal end of the digestive and respiratory tracts, is continually exposed to inhaled and ingested antigens and appears to contribute IgA precursor cells, particularly to the upper respiratory and digestive tracts (4, 27). The unique architecture of the tonsils resembles that of lymph nodes (4) and gut-associated lymphoid tissue (23), in that they have antigen-presenting cells, T and B lymphocytes, IgG- and IgA-containing plasma cells in characteristic regions (4, 23, 46), and deep branched crypts which increase the surface area for trapping environmental materials (4). Supporting evidence for the importance of tonsils in local immune responses include (i) secretion of polymeric IgA in cultured tonsillar cells (28); (ii) the predominance of IgA1, typical of the upper respiratory and digestive tracts (13, 26); and (iii) reduced nasopharyngeal antibody responses to perorally administered live poliovirus in tonsillectomized children and their decreased nasopharyngeal resistance due to diminished secretory IgA levels (37). Although the tonsils were not directly immunized in our study, 5 of the 21 subjects had a history of tonsil surgery. The subjects involved in this study who had had a tonsillectomy and/or adenoidectomy had response patterns and levels of IgA in saliva and nasal wash which were similar to or lower than the average in the other subjects. A more comprehensively designed study comparing the immune responses in individuals with and without a history of tonsil surgery is necessary to determine if responses differ in subjects who have had a tonsillectomy. Alternatively, to determine if tonsils are an IgA induction site for salivary responses, a study in which antigen is topically applied to tonsil tissue is needed and will help determine the potential for tonsils to induce more optimal oral responses.

i.n. immunization of humans with liposomal or free C-GTF antigen of S. mutans resulted in immune responses in nasal secretions, parotid saliva, and serum. The liposomal antigen vaccine induced higher nasal but similar salivary IgA responses, compared to responses induced with the free antigen vaccine. Additional studies are needed to identify ways of inducing predominantly salivary IgA responses, in order to design a more effective approach to prevent oral diseases, e.g., dental caries.

ACKNOWLEDGMENTS

We thank Jiri Mestecky for myeloma proteins and Christina Jespersgaard from UAB Department of Microbiology for purified cloned glucan binding domain of GTF-I. We also wish to thank Rosie Turner for secretarial help.

This work was supported in part by NIH grants DE09846, DE09081, DE08182, and DE08228 and grants DE06746 from the NIDR.

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