[Computed tomography scan of ovarian tumor].
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Adenocarcinoma↗
Biomedical subjects
S Hamada
Publications and source records attributed to S Hamada.
A plaque assay for assessment of immune responses to Streptococcus mutans cell wall carbohydrate.
A plaque-forming cell (PFC) assay has been developed for measurement of single cell responses to the serotype carbohydrate antigen of the Streptococcus mutans cell wall. Serotype g carbohydrate was purified from a mutanolysin (M1) enzyme digest (and designated M1g) of S. mutans 6715 cell walls by ion exchange and gel filtration chromatography. M1g carbohydrate was esterified by stearoylization (sM1g), and subsequently coated to sheep erythrocytes (sM1g-SRBC). This coating antigen was then used for enumeration of IgM anti-M1g PFC responses from spleens of either mice or rats immunized with S. mutans 6715 antigen. Good splenic IgM anti-M1g PFC responses were seen in either mice or rats given S. mutans whole cells or cell walls, while cell wall lysates or M1g were lowly immunogenic. Of interest was the finding that sM1g induced good IgM anti-M1g PFC responses in mice and in murine spleen cell cultures, in vitro. This study describes a method for assessment of individual antibody-producing cells to a major S. mutans cell wall determinant which should facilitate studies directed to determine mechanisms involved in the induction of immune responses to this important bacterium.
Animals↗
Thyroxine action on the rat liver nuclear thyroid-hormone receptors. Binding of thyroxine to the nuclear non-histone protein and induction of mitochondrial alpha-glycerophosphate dehydrogenase activity.
The possibility that thyroxine (T4) itself exerts the hormonal effect in vivo on the rat liver nuclear receptor was studied with the aid of iopanoic acid (IOP), an inhibitor of the conversion of T4 into tri-iodothyronine (T3). After administration of 2.4 micrograms of T4/100 g body weight to hypothyroid rats for 7 days, T4 and T3 concentrations in serum and in the liver nuclear non-histone protein (NHP) were all increased to the hyperthyroid range. Hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) activity and DNA content increased significantly. The equilibrium association constant (Ka) of the nuclear T3 receptor was unchanged and the maximal binding capacity (Cmax.) increased 1.4-fold. Simultaneous administration of IOP (5 mg/100 g body weight) to the rats given 2.4 micrograms of T4/100 g body weight completely blocked the conversion into T3. The serum T4 was even more increased, whereas the serum T3 decreased to the hypothyroid range. Although the NHP-bound T4 was at a concentration comparable with the rats given T4 alone, no NHP-bound T3 was detected. Yet the alpha-GPD activity was elevated 2.8-fold and the DNA content increased to the same extent as observed in the rats given T4 alone. The Ka and Cmax. of the nuclear receptor were significantly decreased. After administration of 48 or 480 micrograms of T4/100 g body weight for 3 days, serum T4 and T3 were markedly increased. The NHP-bound T3 was also increased, but no NHP-bound T4 was detected. The alpha-GPD activity was markedly elevated, but the DNA content was unchanged. The Cmax. per g of liver was increased, whereas the Ka remained unchanged. Simultaneous administration of IOP to these animals could not completely block the T4 conversion. The observed hormonal effects in the absence of nuclear T3 indicate that T4 possesses the intrinsic hormonal activities on the rat liver. T4 is less potent in induction of alpha-GPD activity but as potent in increment of hepatic DNA as T3. Although the binding site for T4 is not fully characterized, it appears to be acidic NHP. T4 is an active hormone, yet is also a prohormone of T3, offering the closest analogy with testosterone.
Animals↗
DAPI staining improved for quantitative cytofluorometry.
DNA-DAPI complexes emit strong bluish white fluorescence when excited by ultraviolet light so that even very small amounts of DNA such as those in mitochondria, chloroplasts, and virus particles can be visualized. Moreover, the staining procedure with DAPI is very simple and requires no hydrolysis. However, DAPI staining was considered unsuitable for quantitative purpose; nonspecific cytoplasmic fluorescence, scattering of strong emission light, and fading of the fluorescence under UV excitation were major problems of DAPI staining in quantitative cytofluorometry. We found that (1) nonspecific cytoplasmic fluorescence could be eliminated by reducing the DAPI concentration to 50 ng/ml, (2) fluorescence decay was markedly decreased by adding electron donors and molecules containing SH radicals in the mounting media, and (3) light scattering became negligible after reducing the intensity of the excitation light. Thus satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained specimens.
Animals↗
An enzyme-linked immunosorbent assay (ELISA) for quantification of antibodies to Streptococcus mutans surface antigens.
An ELISA was developed to quantitate the level of antibodies to various cell surface antigens of the Gram positive bacterium, Streptococcus mutans. Whole cells and purified cell wall components of S. mutans, lipoteichoic acid (LTA) from Streptococcus pyogenes, and dextran T 2000 were employed as coating antigens in this study. Cell walls of S. mutans were purified by mechanical disruption of whole cells followed by differential centrifugation and proteolytic enzyme treatment. Serotype-specific carbohydrate was purified from an autoclaved, lyophilized S. mutans whole cell preparation by column chromatography. LTA was prepared by Sepharose 4B chromatography of a phenol-water extract of S. pyogenes and used for detection of anti-polyglycerophosphate (PGP) antibodies. A rabbit antiserum to S. mutans 6715 (serotype g), which precipitated with purified carbohydrate antigen (RR g), gave good reactions with purified cell walls and whole cells of S. mutans 6715, less activity with RR g and low activity to LTA and dextran when tested by ELISA. Adsorption of this antiserum with whole cells of S. pyogenes resulted in antibody activity with specificity only to the serotype carbohydrate. The specificity of the antibody for homologous coating antigen was RR g greater than cell wall greater than whole cells. An antiserum to S. mutans MT573 (serotype e) contained antibody predominantly to LTA, whereas, anti-S. mutans MT703 (serotype e) reacted with both dextran and LTA; however, the activity to LTA was removed by prior adsorption of the antiserum with S. pyogenes cells. This treatment did not alter the antibody activity to dextran. To establish the sensitivity of ELISA, a purified IgG anti-serotype carbohydrate antibody was prepared by adsorption of anti-S. mutans 6715 antiserum with a mutant of S. mutans which lacks serotype carbohydrate followed by adsorption and elution of specific antibodies from S. mutans 6715 whole cells. The minimum level of sensitivity of ELISA was 12.5 ng of IgG anti-serotype carbohydrate.
Antibodies, Bacterial↗
Oral adjuvants enhance IgA responses to Streptococcus mutans.
The induction of immune responses to orally-administered trinitrophenyl (TNP)-haptenated Streptococcus mutans or its cell wall components and enhancement of immune responses with oral adjuvants has been studied in high IgA responsive C3H/HeJ mice and in gnotobiotic rats. Gastric intubation of TNP-S. mutans to LPS non-responsive C3H/HeJ or syngeneic, LPS responsive C3H/HeN mice induced IgA responses as determined by measuring splenic plaque-forming cell (PFC) responses and IgA anti-TNP antibodies in serum, saliva, and urine. Higher IgA responses always occurred in C3H/HeJ mice given oral S. mutans antigen than similarly treated C3H/HeN animals. Oral administration of the adjuvants concanavalin A or S. mutans cell wall peptidoglycan (PG) with antigen resulted in augmented IgA responses, especially in C3H/HeJ mice. On the other hand, oral administration of muramyl dipeptide (MDP) with antigen boosted anti-TNP responses in C3H/HeN, but not in C3H/HeJ, mice. Gnotobiotic rats given S. mutans whole cells (WC) or purified cell walls (CW) by the oral route exhibited a salivary IgA immune response which was potentiated greater than twofold when antigen was given with PG or MDP. In other studies, S. mutans WC or CW antigen in water-oil-water (W/O/W) emulsion or liposomes was administered by gastric intubation to rats. Significant salivary IgA responses were induced with these antigen-adjuvant preparations. Although rats given S. mutans WC or CW were protected from S. mutans challenge, the greatest degree of caries immunity was obtained in animals which received antigen and adjuvant and which exhibited significant salivary IgA antibody levels. In preliminary studies, it was observed that local injection of rats in the salivary gland region with a ribosomal preparation from S. mutans resulted in a significant salivary IgA response and caries immunity. The potential for soluble and lipid carrier adjuvants in oral vaccines for induction of protective antibodies to S. mutans is discussed.
Acetylmuramyl-Alanyl-Isoglutamine↗
Role of primers in glucan synthesis by glucosyltransferases from Streptococcus mutans strain OMZ176.
The effects of isomaltosaccharides of various molecular weights (isomaltose to dextran T2000) on glucan synthesis by a water-soluble glucan-synthesizing glucosyltransferase enzyme (GTase-S) and a water-insoluble glucan-synthesizing enzyme (GTase-I), both from Streptococcus mutans OMZ176, were examined. The activity of GTase-S was not affected by the addition of the isomaltosaccharides, but GTase-I was stimulated increasingly by isomaltosaccharides with degrees of polymerization more than 10. The GTase-I activity first increased and thereafter decreased slightly with increasing amounts of a soluble dextran. Maximal stimulation occurred at concentrations in the range 0.1 to 0.2 mg ml-1, when dextran T10 was used as a primer. The rate of glucan synthesis was highly enhanced by the combined action of GTase-S and GTase-I. The profile of the net activity of GTase-I in the presence of various amounts of GTase-S was similar to that of GTase-I in the presence of increasing amounts of an exogenous dextran. These results collectively suggest that soluble glucan produced by GTase-S from sucrose acts as an intrinsic primer for the glucan synthesis by GTase-I, indicating the contribution of autopriming in glucan synthesis by crude GTase of S. mutans.
Dextrans↗
Isolation and characterization of the serotype g carbohydrate moiety from an enzyme lysate of Streptococcus mutans 6715 cell walls.
The serotype-specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell-wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo-N-acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C-25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G-100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan--polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A-25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re-chromatographed on a Bio-Gel P-100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti-serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4: 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti-serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an alpha-linked galactose-glucose terminal linkage.
Amino Acids↗
Lymphoid cell responses to bacterial cell wall components: murine B-cell responses to a purified cell wall moiety of Actinomyces.
A mitogenic component, designated fraction C (Fr C), has been purified from a mutanolysin enzyme digest of Actinomyces cell walls by CM Sephadex C-25 ion-exchange and G-100 gel filtration chromatography. Good mitogenic responses were obtained with Fr C over a broad dose range with peak mitogenesis seen with 500 micrograms/culture. Fraction C (mol. wt. = 35,000-40,000) consists of 75% carbohydrate and 23% protein, is non-dialysable, resistant to heat, lysozyme or protease treatment, and partially sensitive to base, and all mitogenic activity is destroyed by either periodate or acid treatment. Fraction C is a B-cell mitogen since it induced responses in nude (nu/nu) and nu/+ BALB/c spleen cell cultures and purified splenic B-cell cultures, but did not stimulate purified splenic T-cell cultures. Similar mitogenic fractions for B cells have been obtained from cell walls of A. naeslundii and from a human isolate of A. viscosus. Good polyclonal IgM synthesis and plaque-forming cell responses to hapten or erythrocytes were obtained in vitro with the purified cell wall fractions derived from all three Actinomyces strains studied. These results indicate that the Actinomyces cell wall possesses a carbohydrate-rich component which activates B cells and may represent a common determinant of this genus.
Actinomyces↗
Non-cariogenicity of the disaccharide palatinose in experimental dental caries of rats.
The caries-inducing activity of palatinose (isomaltulose, alpha-D-glucopyranosyl-1,6-fructose) was examined in in vitro and in vivo experiments, comparing it with other carbohydrates. When Streptococcus mutans was successively subcultured in a broth medium containing 1% palatinose, the strains belonging to serotype a, d, or g did not ferment palatinose, whereas the strains belonging to serotype b, c, e, or f did ferment palatinose. Furthermore, palatinose significantly inhibited the synthesis of insoluble glucan from sucrose by S. mutans. Specific-pathogen-free rats which had been infected with S. mutans 6715 and fed a diet containing 56% palatinose did not develop significant dental caries. However, rats infected similarly, but fed a diet containing sucrose, glucose, fructose, or a glucose-fructose mixture manifested significant caries when compared with the noninfected, sucrose-fed control rats. Furthermore, it was found that replacement of half of the sucrose content with palatinose resulted in decreased caries development compared with caries development in rats fed the sucrose diet.
Animals↗
Purification and immunochemical characterization of Streptococcus sanguis serotype I carbohydrate antigen.
The serotype-specific antigen of Streptococcus sanguis ST3 (serotype I, biotype A) was extracted, chromatographically purified, and characterized by immunological and chemical methods. The antigen was extracted from purified cell walls with hot trichloroacetic acid, followed by ion-exchange chromatography on a DEAE-Sephadex A-25 column and gel filtration through a Sephadex G-100 column. A peak fraction was obtained that gave a single precipitin band when reacted with anti-type I serum. The type I antigen was a polysaccharide composed of glucose, rhamnose, and N-acetylglucosamine in a molar ratio of 1.4:2.5:1.0. Quantitative precipitin inhibition tests with various haptenic sugars indicated that an alpha-glucosidic linkage is the immunodeterminant of the type I antigen.
Chromatography, Gel↗
Effective immunity to dental caries: gastric intubation of Streptococcus mutans whole cells or cell walls induces protective immunity in gnotobiotic rats.
Gnotobiotic rats were given Streptococcus mutans 6715 whole cells (WC), purified cell walls (CW), or cell wall lysate by gastric intubation (GI), and assessments were made of humoral immune responses in serum and saliva and of caries protection. Levels of secretory immunoglobulin A (IgA) and IgG antibodies to S. mutans WC in saliva samples from experimental rats were determined by an enzyme-linked immunosorbent assay. Serum antibody levels of the IgM, IgG, and IgA isotypes were also determined. Similar levels of salivary antibodies were induced in rats given S. mutans WC or CW by GI, whereas lower salivary antibody titers were observed in rats given cell wall lysate by the oral route. The level of serum antibodies in the various groups of rats also reflected the oral antigen used. The specificity of salivary IgA and serum IgG antibodies in the various groups of rats was determined by enzyme-linked immunosorbent assay with lipoteichoic acid, serotype g carbohydrate, dextran, CW, and WC as coating antigens. Salivary IgA and serum IgG antibodies in rats given S. mutans WC or CW by GI were primarily directed to lipoteichoic acid and serotype g carbohydrate. The presence of salivary IgA antibodies to S. mutans in rats given either S. mutans WC or CW by GI correlated with a significant reduction in the levels of plaque, numbers of viable S. mutans in plaque, and caries scores when compared with the control animals (infected only). These results demonstrate that particulate antigens of S. mutans induce salivary immune responses when given by GI to gnotobiotic rats and that the presence of these antibodies correlates with caries protection.
Animals↗
Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants.
In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.
Adjuvants, Immunologic↗
Production of monoclonal antibody against a glucosyltransferase of Streptococcus mutans 6715.
A mouse hybrid cell line secreted monoclonal antibody which reacted specifically with Streptococcus mutans 6715 (serotype g) glucosyltransferase (GTase)-synthesizing water-insoluble glucan and inhibited with enzyme reaction. The antibody was cross-reactive with GTase of serotype d but not with GTase of other serotypes of S. mutans when an enzyme-linked immunosorbent assay was used.
Animals↗
Purification and immunochemical characterization of Streptococcus sanguis ATCC 10557 serotype II carbohydrate antigen.
Cell wall carbohydrate antigen of Streptococcus sanguis ATCC 10557 (serotype II/biotype B) was extracted from purified cell walls by treatment with 5% trichloroacetic acid at 4 degrees C for 8 h. The extract was purified by chromatography on DEAE-Sephadex A-25 and Sephadex G-100 columns. The purified carbohydrate antigen produced a single precipitin band against anti-type II serum, which fused with the band produced by the autoclaved extract or the phenol-water extract of the S. sanguis cells. The type II antigen was a polysaccharide composed of glucose, galactose, rhamnose, and N-acetylgalactosamine in a molar ratio of approximately 3:6:3:2. Quantitative precipitin inhibition tests with various haptenic sugars indicated that N-acetylgalactosamine was a major determinant of the type II antigen.
Amino Acids↗
Increases in brain nuclear triiodothyronine receptors associated with increased triiodothyronine in hyperthyroid and hypothyroid rats.
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Animals↗
Cariostatic effect of palatinose on experimental dental caries in rats.
Specific pathogen-free Sprague-Dawley rats infected with Streptococcus mutans MT8148R (serotype c) developed a severe dental caries when fed a diet containing 56% sucrose (diet #2000). Complete replacement of the dietary sucrose with palatinose, a structural isomer of sucrose, however, resulted in negligible caries induction and plaque accumulation. Replacement of half of the sucrose content of diet #2000 with palatinose induced dental caries; however, the caries score was significantly lower than that induced by diet #2000. No significant reduction in caries development was observed when a quarter of the dietary sucrose was replaced with palatinose.
Animals↗
Characterization of a mutant of serotype g Streptococcus mutans strain 6715 lacking dextran-induced agglutination.
A spontaneous mutant of Streptococcus mutans 6715 (serotype g) defective in dextran-induced agglutination ability was isolated. The wild type strain and its mutant were termed as 6715-DP and 6715-DN, respectively. Both strains possessed serotype g antigen, and exerted similar sugar fermentation patterns. Strain 6715-DP was rapidly and strongly agglutinated upon addition of high molecular weight dextran, whereas the mutant strain 6715-DN was not. [14C]Dextran prepared from Leuconostoc mesenteroides dextransucrase and [14C]sucrose bound to fresh or lyophilized 6715-DP cells, but not to the mutant 6715-DN cells. However, both strains adhered to a glass surface in the presence of sucrose. Furthermore, heat-treated (100 degrees C, 10 min) cells of both strains bound cell-free glucosyltransferase, although dextran agglutination ability of strain 6715-DP was destroyed by this treatment, indicating that receptors for dextran and glucosyltransferase were different entities. Furthermore, serotype c, e, and f strains S. mutans did not agglutinate upon addition of dextran, nor did they bind [14C]dextran. However, all these strains and both 6715-DP and 6715-DN strains induced marked dental caries in SPF rats. It is concluded that dextran-induced agglutination ability is not a necessary condition for S. mutans to induce dental caries.
Agglutination↗