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Relationship of Treponema pallidum to Acidic Mucopolysaccharides T. J. FITZGERALD,* R. C. JOHNSON, AND D. M. RITZI Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Received for publication 26 December 1978
Attempts were made to relate Treponema pallidum to the acidic mucopolysaccharides that occur in vivo within host ground substance and in vitro on the surface of cultured testicular cells. Infected testicular tissue was fixed and processed for transmission electron microscopy in the presence of ruthenium red. The use of this inorganic dye demonstrated the large quantity of mucopolysaccharide within testicular tissue and the intimate association of treponemes with this material. Wheat germ agglutinin and soybean agglutinin agglutinated freshly harvested trypsinized testicular cells and trypsinized cultured cells derived from normal rabbit testes (NRT). When stained with toluidine blue, both cell preparations were metachromatic. Prior treatment of cultured NRT cells with hyaluronidase slightly decreased their sensitivity to agglutination by wheat germ agglutinin and soybean agglutinin. Lectin agglutination, metachromasia, and hyaluronidase susceptibility indicated that freshly harvested testicular cells and NRT cells have surface-associated acidic mucopolysaccharides that are probably hyaluronic acid and chondroitin sulfate. A rabbit erythrocyte "sandwich" technique was devised to show that hyaluronidase removed wheat germ agglutinin receptors from the cultured NRT cells. Prior incubation of NRT cells with hyaluronidase, followed by the addition of T. pallidum, resulted in a reduction in numbers of treponemes attached to the NRT cells. The attachment of T. pallidum appears to be mediated through the acidic mucopolysaccharides on the surface of NRT cells. The findings are discussed in terms of the importance of host ground substance mucopolysaccharide to the syphilitic infective process.
Syphilis can be described as a generalized substance is found within all tissues. In relative infection. Within hours after inoculation of rab- terms, large amounts occur within testicular and bits, Treponema pallidum can be detected in dermal tissues (5, 19). Furthermore, ground sublow numbers in almost every organ and tissue. stance provides structural integrity for blood This also appears to be true after transmission and lymphatic vessels. Thus, within tissues large and entry of the organisms into humans. T. amounts of ground substance are found perivaspallidum, however, preferentially multiplies cularly (1, 5, 7, 13, 20-22). The purpose of this paper was to relate T. only within certain tissues, thereby producing clinical manifestations. In rabbit syphilis, opti- pallidum to the mucopolysaccharides that are mal treponemal multiplication occurs after in- present in vivo within ground substance and in oculation of the testis and the dermis; far less vitro on the surface of cultured cells. multiplication occurs when other tissues are inMATERIALS AND METHODS oculated. In addition, histological sections have indicated that large numbers of organisms localT. pallidunL The organisms were maintained and ize perivascularly within infected tissue (9, 14, harvested in tissue culture medium as previously described (10). 24, 25, 27, 30). Transmission electron microscopy. Pieces of In attempting to explain generalized infection, testicular tissue (1 mm3) were fixed for 2 to 4 preferential multiplication, and perivascular lo- infected h at room temperature with 3% glutaraldehyde in 0.1 T. denominator one common emerges. cation, cacodylate buffer, washed three times with cacopallidum may be strictly dependent on the pres- M dylate buffer, and postfixed in 1% osmium tetroxide ence of ground substance, the intercellular "ce- for 5 h. The samples were then stained en bloc with ment" between tissue cells. This material is com- 1% aqueous uranyl acetate, dehydrated through a prised of proteins complexed with acidic muco- graded ethanol series, and embedded in Epon. For polysaccharides, which are predominantly hy- samples exposed to ruthenium red, the inorganic dye aluronic acid and chondroitin sulfate. Ground was included in both fixatives at a concentration of 252
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700 [ig/ml. The embedded tissue was cut with a diamond knife on an LKB ultratome microtome, and the sections were mounted on Formvar-carbon-coated grids. All sections were stained with 2% uranyl acetate followed by lead citrate and examined in a Siemens Elmiskop I electron microscope. Testicular cells. Testes were removed from an uninfected rabbit and cut into small pieces. These were added to trypsin (0.25%, wt/vol)-ethylenediaminetetraacetic acid (0.01%, wt/vol) in phosphatebuffered saline at pH 7.8. After 20 min, the cells were centrifuged at 1,000 x g at 40C for 10 min. The supernatant fluid was discarded, and the cell pellet was suspended in tissue culture medium (10) without dithiothreitol, glutathione, or cysteine. After settling for 5 min, the top half of the cell suspension was removed for lectin agglutination experiments. This preparation is termed freshly harvested testicular cells. Cultured mammalian cells. Primary cultures were established from normal rabbit testes (NRT) as previously described (12). These cell cultures were grown to confluency and passaged in vitro two to four times. For lectin agglutination experiments, trypsinethylenediaminetetraacetic acid was added to the confluent monolayers for 10 min. The cells were centrifuged at 1,000 x g at 40C for 10 min. The supernatant fluid was discarded, and the cell pellet was suspended in tissue culture medium (10) without dithiothreitol, glutathione, or cysteine. This preparation is termed cultured NRT cells. Plant lectins. Wheat germ agglutinin (WGA) and soybean agglutinin (SBA) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Lectins were dissolved in physiological saline at twice their intended final concentrations. For agglutination experiments, 0.5 volume of each lectin solution was added to 0.5 volume of test suspension. Wherever agglutination was observed, specific inhibition was demonstrated by adding 5 mg of N-acetyl-D-glucosamine per ml to neutralize WGA agglutination, or 5 mg of N-acetyl-D-galactosamine per ml to neutralize SBA agglutination. The specificity of WGA agglutination was tested by adding 5 mg of glucose, lactose, sucrose, and N-acetyl-D-galactosamine per ml. The specificity of SBA agglutination was tested by adding 5 mg of glucose, lactose, sucrose, and N-acetyl-D-glucosamine per ml. Hyaluronidase. Hyaluronidase type IV derived from bovine testes with a specific activity of 1,200 National Federation units per mg was obtained from Sigma Chemical Co. Hyaluronidase derived from Streptomyces hyalurolyticus with a specific activity of 2,000 turbidity-reducing units per mg of protein was obtained from Miles Laboratories (Elkhart, Ind.). Incubations were performed for 18 h due to the relatively slow reactivity of hyaluronidase (5). RBC agglutination. Bovine hyaluronidase (1,200 National Federation units per ml) and streptomyces hyaluronidase (3 turbidity-reducing units per ml) in tissue culture medium without fetal bovine serum were added to cultured NRT cells in Sykes-Moore chambers (26). Tissue culture medium without fetal bovine serum was added to control chambers. After 18 h at 36°C, the enzymes and the control medium were removed by flushing the chambers with 5 volumes of
253
culture medium. WGA at 250 jig/ml was added for 1 h, then removed. The chambers were flushed with 5 volumes of culture medium. Rabbit erythrocytes (RBC) at 5 x 107 per ml were then added to the chambers. After 30 min, the chambers were flushed with 5 volumes of culture medium. Counting of treponemes. Cultured NRT cells were grown to 20% confluency in Sykes-Moore chambers. Bovine hyaluronidase (1,200 National Federation units per ml) and streptomyces hyaluronidase (3 turbidity-reducing units per ml) in tissue culture medium without fetal bovine serum were added. Control chambers received only the culture medium. After 18 h at 36°C, the enzymes and control medium were removed by flushing the chambers with 5 volumes of culture medium. Freshly harvested preparations of T. pallidum were then added. At various intervals, the number of attached treponemes per cultured cell was estimated after observing approximately 100 cultured cells. RESULTS
Ground substance, the intercellular material between tissue cells, is difficult to visualize by using routine methods of fixation and staining. Rabbits were infected intratesticularly with T. pallidum, and after 10 days the animals were sacrificed and the testes were removed. Small pieces of tissue were immediately placed into glutaraldehyde and processed for transmission electron microscopy. Figures 1A and 2A show infected tissue at different magnifications. The membranes and cytoplasmic constituents of the tissue cells were deeply stained. In contrast, very little electron-dense material was observed between the tissue cells. Numerous treponemes are visible in Fig. 2A, and the association of organisms with intercellular material is minimal. Only a slight amount of electron-dense material is apparent immediately surrounding the organisms. Ruthenium red reacts with acidic mucopolysaccharides within ground substance (21). When identical preparations of infected testes were fixed and processed with this inorganic dye, the visualization of the ground substance was greatly enhanced. This also produced an intense staining of the tissue cells. To differentiate the intercellular material more clearly, fixation and processing were performed without the uranyl acetate stain. As shown in Fig. 1B and 2B, this altered procedure diminished the staining of the tissue cells, thereby enhancing the visualization of the ruthenium red ground substance. In contrast to the minimal electron-dense intercellular material in Fig. 1A and 2A, a much heavier electrondense intercellular material was apparent. The involvement of treponemes with ground substance is far more obvious in Fig. 2B than in Fig.
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2A. The treponemes were deeply enmeshed within this material. Syphilitic lesions contain a mucoid substance that has been identified as hyaluronic acid and chondroitin sulfate (24, 25, 28-30). Plant lectins
were used to determine the presence of these two acidic mucopolysaccharides. WGA agglutinates compounds containing N-acetyl-D-glucosamine, a major component of hyaluronic acid;
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(arrows). 255
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FITZGERALD, JOHNSON, AND RITZI
INFECT. IMMUN.
coincubation with various sugars. N-Acetyl-Dglucosamine prevented the precipitate, whereas N-acetyl-D-galactosamine, sucrose, glucose, and lactose did not prevent the precipitate. The previous studies had shown that cultured NRT cells contained surface receptors that reacted with WGA. Attempts were made to remove these receptors enzymatically with hyaluronidase without prior treatment of the cells with trypsin. A procedure was devised involving an RBC "sandwich" technique, which was based on the fact that RBC also contain surface WGA receptors (5). WGA was incubated with cultured cells growing in Sykes-Moore chambers. After washing to remove unattached WGA, rabbit RBC were added to the chambers. These RBC then reacted with the WGA attached to the cultured cells, resulting in binding of RBC to the cultured cells. Hyaluronidase effectively removed a large portion of the WGA receptors on the cultured cells. Figure 4A shows cultured cells incubated with WGA, then with RBC. Numerous RBC attached to the cultured cells, indicating the presence of WGA receptors. Figure 4B shows cultured cells preincubated with hyaluronidase, then incubated with WGA, followed by RBC. Far fewer RBC were attached to the cultured cells, indicating a marked reduction in WGA receptors. Figure 4C represents a control preparation consisting of cultured cells incubated only with RBC. In this preparation the RBC did not attach to the cultured cells. The specificity of this WGA reaction was tested by adding Nacetyl-D-glucosamine to the cultured cells at the same time as the WGA. This was followed by washing and then addition of RBC. As expected, the RBC failed to attach to the cultured cells. This procedure established that hyaluronidase effectively removed WGA receptors that were probably mucopolysaccharides. The last series of experiments determined whether the removal of the mucopolysaccharides affected the cultured cells. TABLE 1. Agglutination of cells by WGA and SBAa attachment of T. pallidum to thewith bovine and Cultured cells were incubated the After removing hyaluronidase. streptomyces Freshly harvested cells Cultured NRT Lectin testicular cells concn enzymes by washing with tissue culture medium, (Ag/ml) freshly harvested preparations of T. pallidum SBA WGA SBA WGA were added to the hyaluronidase-treated cul+++ +++ +++ +++ 500 tured cells and to cultured cells not treated with +++ ++ ++ +++ 250 hyaluronidase. The number of organisms at++ ++ +++ +++ 125 tached per cultured cell was observed during ++ + + ++ 62 incubation at 300C in an atmosphere of 5% car++ + ++ 31 _ + + 15 bon dioxide-92.5% nitrogen-2.5% oxygen. This + 8 experiment was performed four times with iden4 _ _ tical results. The data were not summarized 0 _ _ because different concentrations of treponemal +++, Maximum agglutination; +, minimum agglu- preparations were used. The data from a representative experiment with an initial inoculum of tination; -, no agglutination.
tyl-D-galactosamine, a major component of chondroitin sulfate. Trypsinized cell suspensions were prepared from freshly harvested testicular cells and cultured NRT cells. Both cell preparations were adjusted to approximately 8 x 106 cells per ml and then incubated with various concentrations of each lectin for 3 h at room temperature. As indicated in Table 1, each lectin agglutinated the freshly harvested testicular cells and cultured NRT cells. With the higher concentrations, dense mats of agglutinated cells were detected. The minimal concentration of lectins producing agglutination was similar for both types of cells. In addition, prior incubation with hyaluronidase slightly decreased the sensitivity of the cultured NRT cells to agglutination by the lectins. The specificity of these reactions was determined by adding different sugars to the reaction mixtures. The agglutination produced by WGA was inhibited by addition of N-acetyl-D-glucosamine, but not by N-acetyl-D-galactosamine, sucrose, glucose, or lactose. The agglutination produced by SBA was inhibited by addition of N-acetyl-D-galactosamine, but not by N-acetylD-glucosamine, sucrose, glucose, or lactose. Addition of the higher concentrations of WGA resulted in a precipitate at the surface of freshly harvested testicular cells and cultured NRT cells. This precipitate was observed within minutes after addition of WGA. Figure 3B shows cultured NRT cells in the presence of saline, and Fig. 3A shows cultured NRT cells in the presence of WGA at 250 [g/ml. This precipitate exhibited metachromasia when stained with toluidine blue (0.5%). When the cultured cells were preincubated with hyaluronidase and then exposed to WGA, far less precipitate was detected, and correspondingly less metachromasia was associated with the cells. The specificity of this WGA-induced precipitation was determined by
-
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257
FIG. 3. (A) Cultured NRT cells incubated with 250 pg of WGA per ml in saline. (B) Cultured NRT cells incubated with saline. Bar, 8 um.
5 x 107 treponemes per ml are shown in Table 2. During the initial 43 h, approximately half as many treponemes were attached to the cultured cells treated with hyaluronidase. During this period, the percentage of motile treponemes
within both chambers was almost identical. Beyond 43 h, similar numbers of treponemes were attached within each preparation. This may have been due to the better retention of motility within the hyaluronidase-treated chambers.
258
FITZGERALD, JOHNSON, AND RITZI
INFECT. IMMUN.
.S.
FIG. 4. (A) CutrdNTclsincubated with 250 p~gof WGA per ml, followed by addition of rabbit RBC. Arrow indicates RBC. (B) Cultured NRT cells pretreated with hyaluronidase, then incubated with 250 pg of WGA per ml, followed by addition of RBC. Arrow indicates RBC. (C) Cultured NRT cells incubated with RBC. Arrow indicates RBC. Bar, 40 jim.
TABLE 2. Pretreatment of cultured NRT cells with hyaluronidase and subsequent effects on treponemal attachment Treponemes per cell Time
(h)
Control without
hyalu-
ronidase
1 4 6 19 43 74 91 120
2-4 15-20 20-30 20-60 20-40 5-40 5-25 0-10
Hyl
HyaUronidase treated teed 1 3-5 10-15 10-40 10-20 15-25 5-20 1-5
% Motility
Control without .
hyalu-
ronidase 100 100 90 96 92 75 25 10
Hyaluronidase treated trae
100 100 100 90 92 100 80 20
DISCUSSION One aim of this research was to relate T. pallidum to ground substance, the intercellular cement that provides structural integrity within most tissue (1, 5, 7, 13, 20-22). This material is a highly polymerized, gel-like mixture of water, electrolytes, metabolites, dissolved gases, trace elements, vitamins, enzymes, carbohydrates, and lipids (8). The two major constituents are proteins and various types of acidic mucopolysaccharides. Hyaluronic acid, the predominant mucopolysaccharide, appears to be universally distributed within ground substance (1). In relation to other tissues of the body, the testis contains large amounts of ground substance (5, 19). Electron micrographs of ruthe-
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259
nium red-stained infected testicular tissue dem- surface of the cultured cell is acidic mucopolyonstrated the prevalence of mucopolysaccharide saccharide. The application of these findings to the syphbetween the tissue cells. Inasmuch as syphilis is primarily an extracellular infection (9, 14, 24, 25, ilitic infective process points to ground sub27, 30), the treponemes should be intimately stance as an important host tissue constituent associated with the ground substance. This was for T. pallidum. readily apparent in the micrographs. There are three in vivo observations that imAdditional indicators of mucopolysaccharides ply a direct involvement of the treponemes with within testicular tissue were provided by the ground substance material. First, syphilis can be lectin experiments and by the metachromatic described as a generalized infection. Within observations. Freshly harvested rabbit testicular hours after the initial entry into the host, the cells were agglutinated by WGA and SBA. WGA organisms disseminate and are found in low reacts with receptors containing N-acetyl-D-glu- numbers in almost every tissue of the body. It cosamine, a major constituent of hyaluronic has been postulated by Fitzgerald et al. (11) that acid; SBA reacts with receptors containing N- attachment to tissue cells is a virulence deteracetyl-D-galactosamine, a major constituent of minant of T. pallidum, and that attachment is chondroitin sulfate. The testicular cells after an important step in the disease process. T. staining with toluidine blue were metachro- pallidum (Nichols strain) is capable of attaching matic, a characteristic of acidic mucopolysaccha- to a wide variety of cultured cell types including rides (2-4). those derived from testis, skin, cervix, kidney, A second aim of this research was to establish spleen, lung, urethra, and nerve tissue (11). Asan in vitro relationship between T. pallidum and suming that the organisms attach in vivo, as acidic mucopolysaccharides. Various cultured they do in vitro to cultured cells, the attachment mammalian cells synthesize acidic mucopolysac- sites would have to consist of a substance that is charides during in vitro growth (6, 15-17, 23, 31). generally distributed throughout the body. The cultured NRT cells contained receptors that Ground substance mucopolysaccharides are uniwere agglutinated by both WGA and SBA. This versal constituents of mammalian tissue cells. It was demonstrated with trypsinized cells in sus- is conceivable that the treponemes attach to pension and with growing cells within culture various tissues through these mucopolysacchavessels. The minimum quantity of each lectin rides. A second in vivo observation relates T. pallirequired for agglutination was slightly increased by prior incubation with hyaluronidase, which dum to ground substance. Within the infected degrades both hyaluronic acid and chondroitin host, although organisms are distributed sulfate. Another indicator that the NRT cells throughout the body, they preferentially multicontained acidic mucopolysaccharides was the ply within specific tissues. In rabbit syphilis, metachromasia after staining with toluidine extensive multiplication occurs within dermal and testicular tissues. Temperature appears to blue. The interaction of trypsinized cultured cells play an important role (18). The presence of with high concentrations of WGA resulted in a ground substance may also be important. Virprecipitate at the surface of the cells. When the tually all tissues contain this material. Certain cultured cells were incubated with hyaluroni- tissues, however, such as the testis (5, 19) and dase, far less precipitate was detected after add- the dermis (5), have higher concentrations of ing the WGA. The precipitate, which was met- mucopolysaccharides. In addition, the placenta, achromatic, was probably acidic mucopolysac- the umbilical cord, the aorta, and the eye also have high concentrations (5). Clinical manifescharide. T. pallidum (Nichols strain) attaches to cul- tations in syphilis may occur within all of these tured mammalian cells. There are two surface tissues. These manifestations are attributed to receptor sites to consider in this attachment multiplication of T. pallidum. Thus, the orgaphenomenon: (i) the receptor site on the cul- nisms appear to exhibit a predilection for multured mammalian cell to which the organism tiplication within tissues that contain high conattaches, and (ii) the receptor site on T. palli- centrations of acidic mucopolysaccharides. A third observation relates T. pallidum to dum that interacts with the cultured cell surface. Hyaluronidase treatment removed some of the ground substance. Since structural integrity outer layer of mucopolysaccharide from the sur- within tissues is provided by ground substance, face of the cultured cells. This was shown by the large amounts are present immediately surRBC sandwich technique. The subsequent ad- rounding blood and lymphatic vessels (1, 5, 7, dition of treponemes resulted in far fewer at- 13, 20-22). Histological sections of infected tistached organisms per cultured cell. This obser- sues have demonstrated that T. pallidum tends vation suggests that the receptor site on the to occur perivascularly (9, 14, 24, 25, 27, 30).
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FITZGERALD, JOHNSON, AND RITZI
Thus, within specifically infected tissues, the organisms localize in areas that contain relatively large amounts of acidic mucopolysaccharides. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Elizabeth Thompson Wolff in performing these experiments and Tim Leonard in processing and printing the photomicrographs. This investigation was supported by Public Health Service grants AI-08124 and AI-12978 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Balazs, E. A. 1970. Chemistry and molecular biology of the intercellular matrix, vol. 3. Academic Press, New York. 2. Belanger, L. F., and A. Hartnett. 1960. Persistent toluidine blue metachromasia. J. Histochem. 8:75. 3. Belanger, L. F., and B. B. Migicovsky. 1961. A comparison between different mucopolysaccharide stains as applied to chick epiphyseal cartilage. J. Histochem. 9: 73-86. 4. Blumenkrantz, N. 1957. Microtest for mucopolysaccharides by means of toluidine blue. Clin. Chem. 3:696702. 5. Brimacombe, J. S., and J. M. Weber. 1964. Mucopolysaccharides, chemical structure, distribution, and isolation. Elsevier Publishing, Amsterdam. 6. Burger, M. M., and G. S. Martin. 1972. Agglutination of cells transformed by rous sarcoma virus by wheat germ agglutinin and concanavalin A. Nature (London) New Biol. 237:9-12. 7. Cameron, E. 1966. Hyaluronidase and cancer. Pergamon Press, Oxford. 8. Cameron, E., and L. Pauling. 1973. Ascorbic acid and the glucosaminoglycans. Oncology 27:181-192. 9. DeLamater, E. D., V. R. Saurino, and F. Urbach. 1952. Studies on the immunology of spirochetoses. I. Effect of cortisone on experimental spirochetosis. Am. J. Syph. 36:127-139. 10. Fitzgerald, T. J., and R. C. Johnson. 1979. Surface mucopolysaccharides of Treponema pallidum. Infect. Immun. 24:244-251. 11. Fitzgerald, T. J., R. C. Johnson, J. N. Miller, and J. A. Sykes. 1977. Characterization of the attachment of Treponemapallidum (Nichols strain) to cultured mammalian cells and the potential relationship of attachment to pathogenicity. Infect. Immun. 18:467-478. 12. Fitzgerald, T. J., J. N. Miller, and J. A. Sykes. 1975. Treponema pallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival. Infect. Immun. 11:1133-1140.
13. Gersh, I., and H. R. Catchpole. 1949. The organization of ground substance and basement membrane and its significance in tissue injury, disease, and growth. Am. J. Anat. 85:457-521. 14. Gregoriew, P. S., and K. G. Jarisheva. 1928. The histological structure of syphilitic lesions of rabbits. Am. J. Syph. 12:67-81. 15. Groasfield, H. 1957. Positive mucin clot test in supernates of cultures of avian embryonic brain. Proc. Soc. Exp. Biol. Med. 96:844-846. 16. Groasfield, H. 1958. Studies of production of hyaluronic acid in tissue culture. The presence of hyaluronidase in embryo extract. Exp. Cell Res. 14:213-216. 17. Grossfield, H., K. Meyer, G. Godman, and A. Linker. 1957. Mucopolysaccharides produced in tissue culture. J. Cell Biol. 3:391-396. 18. Hollander, D. H., and T. B. Turner. 1954. The role of temperature in experimental syphilis infection. Am. J. Syph. 38:489-505. 19. Johnson, A. D., W. R. Gomes, and N. L. Vandemark. 1970. The testis, vol. 1. Academic Press, New York. 20. Luft, J. H. 1966. Fine structure of capillary and endocapillary layer as revealed by ruthenium red. Fed. Proc. 25:1773-1783. 21. Luft, J. H. 1971. Ruthenium red and violet. II. Fine structural localization in animal tissues. Anat. Rec. 171:
369-416. 22. Moore, R. D., and M. D. Schoenberg. 1957. Studies on connective tissue. I. The polysaccharides of thekuman umbilical cord. Arch. Pathol. 64:39-45. 23. Morris, C. C. 1960. Quantitative studies on the production of acid mucopolysaccharides by replicate cell cultures of rat fibroblasts. Ann. N.Y. Acad. Sci. 86:878-915. 24. Scott, V., and G. J. Dammin. 1950. Hyaluronidase and experimental syphilis. III. Metachromasia in syphilitic orchitis and its relation to hyaluronic acid. Am. J. Syph. 34:501-514. 25. Scott, V., and G. J. Dammin. 1954. Morphologic and histochemical sequences in syphilitic and tuberculous orchitis in the rabbit. Am. J. Syph. 38:189-202. 26. Sykes, J. A., and E. B. Moore. 1960. A simple tissue culture chamber. Tex. Rep. Biol. Med. 18:288-297. 27. Turner, D. R., and D. J. M. Wright. 1973. Lymphadenopathy in early syphilis. J. Pathol. 110:305-308. 28. Turner, T. B. 1970. Syphilis and the treponematoses, p. 346-390. In S. Mudd (ed.), Infectious agents and host reactions. W. B. Saunders, Philadelphia. 29. Turner, T. B., and D. H. Hollander. 1950. Cortisone in experimental syphilis. Johns Hopkins Hosp. Bull. 87: 505-509. 30. Turner, T. B., and D. H. Hollander. 1954. Studies on the mechanism of action of cortisone in experimental syphilis. Am. J. Syph. 38:371-387. 31. Wright, T. N., and R. Ross. 1975. Proteoglycans in primary arteries. II. Synthesis and secretion of glycosaminoglycans by arterial smooth muscle cells in culture. J. Cell Biol. 67:660-674.
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Relationship of Treponema pallidum to Acidic Mucopolysaccharides T. J. FITZGERALD,* R. C. JOHNSON, AND D. M. RITZI Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Received for publication 26 December 1978
Attempts were made to relate Treponema pallidum to the acidic mucopolysaccharides that occur in vivo within host ground substance and in vitro on the surface of cultured testicular cells. Infected testicular tissue was fixed and processed for transmission electron microscopy in the presence of ruthenium red. The use of this inorganic dye demonstrated the large quantity of mucopolysaccharide within testicular tissue and the intimate association of treponemes with this material. Wheat germ agglutinin and soybean agglutinin agglutinated freshly harvested trypsinized testicular cells and trypsinized cultured cells derived from normal rabbit testes (NRT). When stained with toluidine blue, both cell preparations were metachromatic. Prior treatment of cultured NRT cells with hyaluronidase slightly decreased their sensitivity to agglutination by wheat germ agglutinin and soybean agglutinin. Lectin agglutination, metachromasia, and hyaluronidase susceptibility indicated that freshly harvested testicular cells and NRT cells have surface-associated acidic mucopolysaccharides that are probably hyaluronic acid and chondroitin sulfate. A rabbit erythrocyte "sandwich" technique was devised to show that hyaluronidase removed wheat germ agglutinin receptors from the cultured NRT cells. Prior incubation of NRT cells with hyaluronidase, followed by the addition of T. pallidum, resulted in a reduction in numbers of treponemes attached to the NRT cells. The attachment of T. pallidum appears to be mediated through the acidic mucopolysaccharides on the surface of NRT cells. The findings are discussed in terms of the importance of host ground substance mucopolysaccharide to the syphilitic infective process.
Syphilis can be described as a generalized substance is found within all tissues. In relative infection. Within hours after inoculation of rab- terms, large amounts occur within testicular and bits, Treponema pallidum can be detected in dermal tissues (5, 19). Furthermore, ground sublow numbers in almost every organ and tissue. stance provides structural integrity for blood This also appears to be true after transmission and lymphatic vessels. Thus, within tissues large and entry of the organisms into humans. T. amounts of ground substance are found perivaspallidum, however, preferentially multiplies cularly (1, 5, 7, 13, 20-22). The purpose of this paper was to relate T. only within certain tissues, thereby producing clinical manifestations. In rabbit syphilis, opti- pallidum to the mucopolysaccharides that are mal treponemal multiplication occurs after in- present in vivo within ground substance and in oculation of the testis and the dermis; far less vitro on the surface of cultured cells. multiplication occurs when other tissues are inMATERIALS AND METHODS oculated. In addition, histological sections have indicated that large numbers of organisms localT. pallidunL The organisms were maintained and ize perivascularly within infected tissue (9, 14, harvested in tissue culture medium as previously described (10). 24, 25, 27, 30). Transmission electron microscopy. Pieces of In attempting to explain generalized infection, testicular tissue (1 mm3) were fixed for 2 to 4 preferential multiplication, and perivascular lo- infected h at room temperature with 3% glutaraldehyde in 0.1 T. denominator one common emerges. cation, cacodylate buffer, washed three times with cacopallidum may be strictly dependent on the pres- M dylate buffer, and postfixed in 1% osmium tetroxide ence of ground substance, the intercellular "ce- for 5 h. The samples were then stained en bloc with ment" between tissue cells. This material is com- 1% aqueous uranyl acetate, dehydrated through a prised of proteins complexed with acidic muco- graded ethanol series, and embedded in Epon. For polysaccharides, which are predominantly hy- samples exposed to ruthenium red, the inorganic dye aluronic acid and chondroitin sulfate. Ground was included in both fixatives at a concentration of 252
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700 [ig/ml. The embedded tissue was cut with a diamond knife on an LKB ultratome microtome, and the sections were mounted on Formvar-carbon-coated grids. All sections were stained with 2% uranyl acetate followed by lead citrate and examined in a Siemens Elmiskop I electron microscope. Testicular cells. Testes were removed from an uninfected rabbit and cut into small pieces. These were added to trypsin (0.25%, wt/vol)-ethylenediaminetetraacetic acid (0.01%, wt/vol) in phosphatebuffered saline at pH 7.8. After 20 min, the cells were centrifuged at 1,000 x g at 40C for 10 min. The supernatant fluid was discarded, and the cell pellet was suspended in tissue culture medium (10) without dithiothreitol, glutathione, or cysteine. After settling for 5 min, the top half of the cell suspension was removed for lectin agglutination experiments. This preparation is termed freshly harvested testicular cells. Cultured mammalian cells. Primary cultures were established from normal rabbit testes (NRT) as previously described (12). These cell cultures were grown to confluency and passaged in vitro two to four times. For lectin agglutination experiments, trypsinethylenediaminetetraacetic acid was added to the confluent monolayers for 10 min. The cells were centrifuged at 1,000 x g at 40C for 10 min. The supernatant fluid was discarded, and the cell pellet was suspended in tissue culture medium (10) without dithiothreitol, glutathione, or cysteine. This preparation is termed cultured NRT cells. Plant lectins. Wheat germ agglutinin (WGA) and soybean agglutinin (SBA) were obtained from Sigma Chemical Co. (St. Louis, Mo.). Lectins were dissolved in physiological saline at twice their intended final concentrations. For agglutination experiments, 0.5 volume of each lectin solution was added to 0.5 volume of test suspension. Wherever agglutination was observed, specific inhibition was demonstrated by adding 5 mg of N-acetyl-D-glucosamine per ml to neutralize WGA agglutination, or 5 mg of N-acetyl-D-galactosamine per ml to neutralize SBA agglutination. The specificity of WGA agglutination was tested by adding 5 mg of glucose, lactose, sucrose, and N-acetyl-D-galactosamine per ml. The specificity of SBA agglutination was tested by adding 5 mg of glucose, lactose, sucrose, and N-acetyl-D-glucosamine per ml. Hyaluronidase. Hyaluronidase type IV derived from bovine testes with a specific activity of 1,200 National Federation units per mg was obtained from Sigma Chemical Co. Hyaluronidase derived from Streptomyces hyalurolyticus with a specific activity of 2,000 turbidity-reducing units per mg of protein was obtained from Miles Laboratories (Elkhart, Ind.). Incubations were performed for 18 h due to the relatively slow reactivity of hyaluronidase (5). RBC agglutination. Bovine hyaluronidase (1,200 National Federation units per ml) and streptomyces hyaluronidase (3 turbidity-reducing units per ml) in tissue culture medium without fetal bovine serum were added to cultured NRT cells in Sykes-Moore chambers (26). Tissue culture medium without fetal bovine serum was added to control chambers. After 18 h at 36°C, the enzymes and the control medium were removed by flushing the chambers with 5 volumes of
253
culture medium. WGA at 250 jig/ml was added for 1 h, then removed. The chambers were flushed with 5 volumes of culture medium. Rabbit erythrocytes (RBC) at 5 x 107 per ml were then added to the chambers. After 30 min, the chambers were flushed with 5 volumes of culture medium. Counting of treponemes. Cultured NRT cells were grown to 20% confluency in Sykes-Moore chambers. Bovine hyaluronidase (1,200 National Federation units per ml) and streptomyces hyaluronidase (3 turbidity-reducing units per ml) in tissue culture medium without fetal bovine serum were added. Control chambers received only the culture medium. After 18 h at 36°C, the enzymes and control medium were removed by flushing the chambers with 5 volumes of culture medium. Freshly harvested preparations of T. pallidum were then added. At various intervals, the number of attached treponemes per cultured cell was estimated after observing approximately 100 cultured cells. RESULTS
Ground substance, the intercellular material between tissue cells, is difficult to visualize by using routine methods of fixation and staining. Rabbits were infected intratesticularly with T. pallidum, and after 10 days the animals were sacrificed and the testes were removed. Small pieces of tissue were immediately placed into glutaraldehyde and processed for transmission electron microscopy. Figures 1A and 2A show infected tissue at different magnifications. The membranes and cytoplasmic constituents of the tissue cells were deeply stained. In contrast, very little electron-dense material was observed between the tissue cells. Numerous treponemes are visible in Fig. 2A, and the association of organisms with intercellular material is minimal. Only a slight amount of electron-dense material is apparent immediately surrounding the organisms. Ruthenium red reacts with acidic mucopolysaccharides within ground substance (21). When identical preparations of infected testes were fixed and processed with this inorganic dye, the visualization of the ground substance was greatly enhanced. This also produced an intense staining of the tissue cells. To differentiate the intercellular material more clearly, fixation and processing were performed without the uranyl acetate stain. As shown in Fig. 1B and 2B, this altered procedure diminished the staining of the tissue cells, thereby enhancing the visualization of the ruthenium red ground substance. In contrast to the minimal electron-dense intercellular material in Fig. 1A and 2A, a much heavier electrondense intercellular material was apparent. The involvement of treponemes with ground substance is far more obvious in Fig. 2B than in Fig.
254
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2A. The treponemes were deeply enmeshed within this material. Syphilitic lesions contain a mucoid substance that has been identified as hyaluronic acid and chondroitin sulfate (24, 25, 28-30). Plant lectins
were used to determine the presence of these two acidic mucopolysaccharides. WGA agglutinates compounds containing N-acetyl-D-glucosamine, a major component of hyaluronic acid;
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(arrows). 255
256
FITZGERALD, JOHNSON, AND RITZI
INFECT. IMMUN.
coincubation with various sugars. N-Acetyl-Dglucosamine prevented the precipitate, whereas N-acetyl-D-galactosamine, sucrose, glucose, and lactose did not prevent the precipitate. The previous studies had shown that cultured NRT cells contained surface receptors that reacted with WGA. Attempts were made to remove these receptors enzymatically with hyaluronidase without prior treatment of the cells with trypsin. A procedure was devised involving an RBC "sandwich" technique, which was based on the fact that RBC also contain surface WGA receptors (5). WGA was incubated with cultured cells growing in Sykes-Moore chambers. After washing to remove unattached WGA, rabbit RBC were added to the chambers. These RBC then reacted with the WGA attached to the cultured cells, resulting in binding of RBC to the cultured cells. Hyaluronidase effectively removed a large portion of the WGA receptors on the cultured cells. Figure 4A shows cultured cells incubated with WGA, then with RBC. Numerous RBC attached to the cultured cells, indicating the presence of WGA receptors. Figure 4B shows cultured cells preincubated with hyaluronidase, then incubated with WGA, followed by RBC. Far fewer RBC were attached to the cultured cells, indicating a marked reduction in WGA receptors. Figure 4C represents a control preparation consisting of cultured cells incubated only with RBC. In this preparation the RBC did not attach to the cultured cells. The specificity of this WGA reaction was tested by adding Nacetyl-D-glucosamine to the cultured cells at the same time as the WGA. This was followed by washing and then addition of RBC. As expected, the RBC failed to attach to the cultured cells. This procedure established that hyaluronidase effectively removed WGA receptors that were probably mucopolysaccharides. The last series of experiments determined whether the removal of the mucopolysaccharides affected the cultured cells. TABLE 1. Agglutination of cells by WGA and SBAa attachment of T. pallidum to thewith bovine and Cultured cells were incubated the After removing hyaluronidase. streptomyces Freshly harvested cells Cultured NRT Lectin testicular cells concn enzymes by washing with tissue culture medium, (Ag/ml) freshly harvested preparations of T. pallidum SBA WGA SBA WGA were added to the hyaluronidase-treated cul+++ +++ +++ +++ 500 tured cells and to cultured cells not treated with +++ ++ ++ +++ 250 hyaluronidase. The number of organisms at++ ++ +++ +++ 125 tached per cultured cell was observed during ++ + + ++ 62 incubation at 300C in an atmosphere of 5% car++ + ++ 31 _ + + 15 bon dioxide-92.5% nitrogen-2.5% oxygen. This + 8 experiment was performed four times with iden4 _ _ tical results. The data were not summarized 0 _ _ because different concentrations of treponemal +++, Maximum agglutination; +, minimum agglu- preparations were used. The data from a representative experiment with an initial inoculum of tination; -, no agglutination.
tyl-D-galactosamine, a major component of chondroitin sulfate. Trypsinized cell suspensions were prepared from freshly harvested testicular cells and cultured NRT cells. Both cell preparations were adjusted to approximately 8 x 106 cells per ml and then incubated with various concentrations of each lectin for 3 h at room temperature. As indicated in Table 1, each lectin agglutinated the freshly harvested testicular cells and cultured NRT cells. With the higher concentrations, dense mats of agglutinated cells were detected. The minimal concentration of lectins producing agglutination was similar for both types of cells. In addition, prior incubation with hyaluronidase slightly decreased the sensitivity of the cultured NRT cells to agglutination by the lectins. The specificity of these reactions was determined by adding different sugars to the reaction mixtures. The agglutination produced by WGA was inhibited by addition of N-acetyl-D-glucosamine, but not by N-acetyl-D-galactosamine, sucrose, glucose, or lactose. The agglutination produced by SBA was inhibited by addition of N-acetyl-D-galactosamine, but not by N-acetylD-glucosamine, sucrose, glucose, or lactose. Addition of the higher concentrations of WGA resulted in a precipitate at the surface of freshly harvested testicular cells and cultured NRT cells. This precipitate was observed within minutes after addition of WGA. Figure 3B shows cultured NRT cells in the presence of saline, and Fig. 3A shows cultured NRT cells in the presence of WGA at 250 [g/ml. This precipitate exhibited metachromasia when stained with toluidine blue (0.5%). When the cultured cells were preincubated with hyaluronidase and then exposed to WGA, far less precipitate was detected, and correspondingly less metachromasia was associated with the cells. The specificity of this WGA-induced precipitation was determined by
-
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257
FIG. 3. (A) Cultured NRT cells incubated with 250 pg of WGA per ml in saline. (B) Cultured NRT cells incubated with saline. Bar, 8 um.
5 x 107 treponemes per ml are shown in Table 2. During the initial 43 h, approximately half as many treponemes were attached to the cultured cells treated with hyaluronidase. During this period, the percentage of motile treponemes
within both chambers was almost identical. Beyond 43 h, similar numbers of treponemes were attached within each preparation. This may have been due to the better retention of motility within the hyaluronidase-treated chambers.
258
FITZGERALD, JOHNSON, AND RITZI
INFECT. IMMUN.
.S.
FIG. 4. (A) CutrdNTclsincubated with 250 p~gof WGA per ml, followed by addition of rabbit RBC. Arrow indicates RBC. (B) Cultured NRT cells pretreated with hyaluronidase, then incubated with 250 pg of WGA per ml, followed by addition of RBC. Arrow indicates RBC. (C) Cultured NRT cells incubated with RBC. Arrow indicates RBC. Bar, 40 jim.
TABLE 2. Pretreatment of cultured NRT cells with hyaluronidase and subsequent effects on treponemal attachment Treponemes per cell Time
(h)
Control without
hyalu-
ronidase
1 4 6 19 43 74 91 120
2-4 15-20 20-30 20-60 20-40 5-40 5-25 0-10
Hyl
HyaUronidase treated teed 1 3-5 10-15 10-40 10-20 15-25 5-20 1-5
% Motility
Control without .
hyalu-
ronidase 100 100 90 96 92 75 25 10
Hyaluronidase treated trae
100 100 100 90 92 100 80 20
DISCUSSION One aim of this research was to relate T. pallidum to ground substance, the intercellular cement that provides structural integrity within most tissue (1, 5, 7, 13, 20-22). This material is a highly polymerized, gel-like mixture of water, electrolytes, metabolites, dissolved gases, trace elements, vitamins, enzymes, carbohydrates, and lipids (8). The two major constituents are proteins and various types of acidic mucopolysaccharides. Hyaluronic acid, the predominant mucopolysaccharide, appears to be universally distributed within ground substance (1). In relation to other tissues of the body, the testis contains large amounts of ground substance (5, 19). Electron micrographs of ruthe-
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259
nium red-stained infected testicular tissue dem- surface of the cultured cell is acidic mucopolyonstrated the prevalence of mucopolysaccharide saccharide. The application of these findings to the syphbetween the tissue cells. Inasmuch as syphilis is primarily an extracellular infection (9, 14, 24, 25, ilitic infective process points to ground sub27, 30), the treponemes should be intimately stance as an important host tissue constituent associated with the ground substance. This was for T. pallidum. readily apparent in the micrographs. There are three in vivo observations that imAdditional indicators of mucopolysaccharides ply a direct involvement of the treponemes with within testicular tissue were provided by the ground substance material. First, syphilis can be lectin experiments and by the metachromatic described as a generalized infection. Within observations. Freshly harvested rabbit testicular hours after the initial entry into the host, the cells were agglutinated by WGA and SBA. WGA organisms disseminate and are found in low reacts with receptors containing N-acetyl-D-glu- numbers in almost every tissue of the body. It cosamine, a major constituent of hyaluronic has been postulated by Fitzgerald et al. (11) that acid; SBA reacts with receptors containing N- attachment to tissue cells is a virulence deteracetyl-D-galactosamine, a major constituent of minant of T. pallidum, and that attachment is chondroitin sulfate. The testicular cells after an important step in the disease process. T. staining with toluidine blue were metachro- pallidum (Nichols strain) is capable of attaching matic, a characteristic of acidic mucopolysaccha- to a wide variety of cultured cell types including rides (2-4). those derived from testis, skin, cervix, kidney, A second aim of this research was to establish spleen, lung, urethra, and nerve tissue (11). Asan in vitro relationship between T. pallidum and suming that the organisms attach in vivo, as acidic mucopolysaccharides. Various cultured they do in vitro to cultured cells, the attachment mammalian cells synthesize acidic mucopolysac- sites would have to consist of a substance that is charides during in vitro growth (6, 15-17, 23, 31). generally distributed throughout the body. The cultured NRT cells contained receptors that Ground substance mucopolysaccharides are uniwere agglutinated by both WGA and SBA. This versal constituents of mammalian tissue cells. It was demonstrated with trypsinized cells in sus- is conceivable that the treponemes attach to pension and with growing cells within culture various tissues through these mucopolysacchavessels. The minimum quantity of each lectin rides. A second in vivo observation relates T. pallirequired for agglutination was slightly increased by prior incubation with hyaluronidase, which dum to ground substance. Within the infected degrades both hyaluronic acid and chondroitin host, although organisms are distributed sulfate. Another indicator that the NRT cells throughout the body, they preferentially multicontained acidic mucopolysaccharides was the ply within specific tissues. In rabbit syphilis, metachromasia after staining with toluidine extensive multiplication occurs within dermal and testicular tissues. Temperature appears to blue. The interaction of trypsinized cultured cells play an important role (18). The presence of with high concentrations of WGA resulted in a ground substance may also be important. Virprecipitate at the surface of the cells. When the tually all tissues contain this material. Certain cultured cells were incubated with hyaluroni- tissues, however, such as the testis (5, 19) and dase, far less precipitate was detected after add- the dermis (5), have higher concentrations of ing the WGA. The precipitate, which was met- mucopolysaccharides. In addition, the placenta, achromatic, was probably acidic mucopolysac- the umbilical cord, the aorta, and the eye also have high concentrations (5). Clinical manifescharide. T. pallidum (Nichols strain) attaches to cul- tations in syphilis may occur within all of these tured mammalian cells. There are two surface tissues. These manifestations are attributed to receptor sites to consider in this attachment multiplication of T. pallidum. Thus, the orgaphenomenon: (i) the receptor site on the cul- nisms appear to exhibit a predilection for multured mammalian cell to which the organism tiplication within tissues that contain high conattaches, and (ii) the receptor site on T. palli- centrations of acidic mucopolysaccharides. A third observation relates T. pallidum to dum that interacts with the cultured cell surface. Hyaluronidase treatment removed some of the ground substance. Since structural integrity outer layer of mucopolysaccharide from the sur- within tissues is provided by ground substance, face of the cultured cells. This was shown by the large amounts are present immediately surRBC sandwich technique. The subsequent ad- rounding blood and lymphatic vessels (1, 5, 7, dition of treponemes resulted in far fewer at- 13, 20-22). Histological sections of infected tistached organisms per cultured cell. This obser- sues have demonstrated that T. pallidum tends vation suggests that the receptor site on the to occur perivascularly (9, 14, 24, 25, 27, 30).
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FITZGERALD, JOHNSON, AND RITZI
Thus, within specifically infected tissues, the organisms localize in areas that contain relatively large amounts of acidic mucopolysaccharides. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Elizabeth Thompson Wolff in performing these experiments and Tim Leonard in processing and printing the photomicrographs. This investigation was supported by Public Health Service grants AI-08124 and AI-12978 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Balazs, E. A. 1970. Chemistry and molecular biology of the intercellular matrix, vol. 3. Academic Press, New York. 2. Belanger, L. F., and A. Hartnett. 1960. Persistent toluidine blue metachromasia. J. Histochem. 8:75. 3. Belanger, L. F., and B. B. Migicovsky. 1961. A comparison between different mucopolysaccharide stains as applied to chick epiphyseal cartilage. J. Histochem. 9: 73-86. 4. Blumenkrantz, N. 1957. Microtest for mucopolysaccharides by means of toluidine blue. Clin. Chem. 3:696702. 5. Brimacombe, J. S., and J. M. Weber. 1964. Mucopolysaccharides, chemical structure, distribution, and isolation. Elsevier Publishing, Amsterdam. 6. Burger, M. M., and G. S. Martin. 1972. Agglutination of cells transformed by rous sarcoma virus by wheat germ agglutinin and concanavalin A. Nature (London) New Biol. 237:9-12. 7. Cameron, E. 1966. Hyaluronidase and cancer. Pergamon Press, Oxford. 8. Cameron, E., and L. Pauling. 1973. Ascorbic acid and the glucosaminoglycans. Oncology 27:181-192. 9. DeLamater, E. D., V. R. Saurino, and F. Urbach. 1952. Studies on the immunology of spirochetoses. I. Effect of cortisone on experimental spirochetosis. Am. J. Syph. 36:127-139. 10. Fitzgerald, T. J., and R. C. Johnson. 1979. Surface mucopolysaccharides of Treponema pallidum. Infect. Immun. 24:244-251. 11. Fitzgerald, T. J., R. C. Johnson, J. N. Miller, and J. A. Sykes. 1977. Characterization of the attachment of Treponemapallidum (Nichols strain) to cultured mammalian cells and the potential relationship of attachment to pathogenicity. Infect. Immun. 18:467-478. 12. Fitzgerald, T. J., J. N. Miller, and J. A. Sykes. 1975. Treponema pallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival. Infect. Immun. 11:1133-1140.
13. Gersh, I., and H. R. Catchpole. 1949. The organization of ground substance and basement membrane and its significance in tissue injury, disease, and growth. Am. J. Anat. 85:457-521. 14. Gregoriew, P. S., and K. G. Jarisheva. 1928. The histological structure of syphilitic lesions of rabbits. Am. J. Syph. 12:67-81. 15. Groasfield, H. 1957. Positive mucin clot test in supernates of cultures of avian embryonic brain. Proc. Soc. Exp. Biol. Med. 96:844-846. 16. Groasfield, H. 1958. Studies of production of hyaluronic acid in tissue culture. The presence of hyaluronidase in embryo extract. Exp. Cell Res. 14:213-216. 17. Grossfield, H., K. Meyer, G. Godman, and A. Linker. 1957. Mucopolysaccharides produced in tissue culture. J. Cell Biol. 3:391-396. 18. Hollander, D. H., and T. B. Turner. 1954. The role of temperature in experimental syphilis infection. Am. J. Syph. 38:489-505. 19. Johnson, A. D., W. R. Gomes, and N. L. Vandemark. 1970. The testis, vol. 1. Academic Press, New York. 20. Luft, J. H. 1966. Fine structure of capillary and endocapillary layer as revealed by ruthenium red. Fed. Proc. 25:1773-1783. 21. Luft, J. H. 1971. Ruthenium red and violet. II. Fine structural localization in animal tissues. Anat. Rec. 171:
369-416. 22. Moore, R. D., and M. D. Schoenberg. 1957. Studies on connective tissue. I. The polysaccharides of thekuman umbilical cord. Arch. Pathol. 64:39-45. 23. Morris, C. C. 1960. Quantitative studies on the production of acid mucopolysaccharides by replicate cell cultures of rat fibroblasts. Ann. N.Y. Acad. Sci. 86:878-915. 24. Scott, V., and G. J. Dammin. 1950. Hyaluronidase and experimental syphilis. III. Metachromasia in syphilitic orchitis and its relation to hyaluronic acid. Am. J. Syph. 34:501-514. 25. Scott, V., and G. J. Dammin. 1954. Morphologic and histochemical sequences in syphilitic and tuberculous orchitis in the rabbit. Am. J. Syph. 38:189-202. 26. Sykes, J. A., and E. B. Moore. 1960. A simple tissue culture chamber. Tex. Rep. Biol. Med. 18:288-297. 27. Turner, D. R., and D. J. M. Wright. 1973. Lymphadenopathy in early syphilis. J. Pathol. 110:305-308. 28. Turner, T. B. 1970. Syphilis and the treponematoses, p. 346-390. In S. Mudd (ed.), Infectious agents and host reactions. W. B. Saunders, Philadelphia. 29. Turner, T. B., and D. H. Hollander. 1950. Cortisone in experimental syphilis. Johns Hopkins Hosp. Bull. 87: 505-509. 30. Turner, T. B., and D. H. Hollander. 1954. Studies on the mechanism of action of cortisone in experimental syphilis. Am. J. Syph. 38:371-387. 31. Wright, T. N., and R. Ross. 1975. Proteoglycans in primary arteries. II. Synthesis and secretion of glycosaminoglycans by arterial smooth muscle cells in culture. J. Cell Biol. 67:660-674.
