Jack D. Zwemer

The Immunohistochemical Localization of Plasma Proteins in Human Gingivae

F LUORESCENT-LABELLED antibodies have found increasing use as highly specific and sensitive histochemical reagents for the detection and discrete localization of infectious agents, injected foreign antigens, and antigenic substances native to the tissues (Coons, A. H.: Internat. Rev. Cytol. 5: 1, 1956). With this immunohistochemical technic, plasma albumin, gamma globulin, and fibrin(ogen) have been demonstrated in blood vessels, lymphatics, and the connective tissue ground substance and interstitial spaces of normal, human tissues (Gitlin, D., Landing, B. H., and Whipple, A.: J. Exper. Med. 97: 163, 1953). In a number of diverse inflammatory pathoses, these plasma proteins exhibit typical patterns of increased tissue localization. Thus, plasma albumin is predominate in non-specifically inflamed tissues; gamma globulin predominates in the lesions of serum sickness and of active rheumatic carditis, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, periarteritis nodosa, and secondary amyloidosis; and fibrin deposits are characteristic of Shwartzman s phenomenon and thrombotic thrombocytopenic purpura (Mellors, R. C., and Ortega, L. G.: Am. J. Path. 32: 455, 1956; Vazquez, J. J., and Dixon, F. J.: Lab. Invest. 6: 205, 1957; and A. M. A. Arch. Path. 66: 504, 1958). In view of the differential localization of albumin, globulin, and fibrin(ogen) in various pathoses and the pathogenetic implications, it seemed desirable to determine the pattern of protein localization in normal and inflamed human gingivae. Technics employed in this study included those of Coons and Kaplan (J. Exper. Med. 91: 1, 1950) together with certain modifications and the controls used by Vazquez and Dixon (J. Exper. Med. 104: 727, 1956). Gamma globulin fractions of rabbit antisera to human albumin, gamma globulin, and fibrinogen (Cutter Laboratories, Berkeley, Calif.) were labelled with fluorescein isothiocyanate according to the method of Marshall, Eveland, and Smith (Proc. Soc. Exper. Biol. & Med. 98: 898, 1958). Gingival biopsies from 10 clinically normal subjects and from 10 patients presenting for dental extractions, with moderate to advanced inflammatory periodontal disease, were secured by the technic of Ziskin, Loughlin and Siegel (Am. J. Orthodont. & Oral Surg. 30: 758, 1944). The frozen tissue specimens were stabilized for cutting on previously moistened cellophane dialysis film in a modification of the method of Bush and Hewitt (Am. J. Path. 28: 863, 1952). Cetyl alcohol (0.25 per cent w/v), added to either the ethanol-ether or 95 per cent ethanol fixatives, facilitated adhesion of the sections to the slides. The appropriately stained and mounted sections were examined microscopically for specific fluorescence. Visual comparisons were made of the intensity ofstaining. Both normal and inflamed gingival tissues possessed the geographic distribution of plasma proteins reported by Gitlin and associates for the tongue and skin of normal subjects. In both normal and inflamed tissues, albumin was the consistently predominant plasma protein, with gamma globulin and fibrin(ogen) also present in significant but distinctly lesser amounts. Fluorescence associated with fibrin(ogen) often approached, and occasionally exceeded, that observed with gamma globulin. Inflamed tissue exhibited no conspicuous relative increase in either globulin or fibrin(ogen) and therefore the histochemical evidence in advanced inflammatory periodontal disease is not reminiscent of the patterns found in either serum sickness or Shwartzmans phenomenon or their presumed pathogenetic congeners. Further studies should be made of the localization of plasma proteins in the periodontal membrane and alveolar bone.

The immunohistochemical localization of plasma albumin in murine dentinal tissue.

V LUORESCENT-LABELED antibodies have been used to localize plasma proteins in the Cells and interstitial spaces of vascular connective tissues in man and animals (Zwemer, J. D., Meng, H. M., and Nutter, R. L.: J. D. Res. 38: 844, 1959). One report has also described the distribution of plasma albumin and y globulin in the lacunar cells of human tracheal cartilage (Gitlin, D., Landing, B. H., and Whipple, A.: J. Exper. Med. 97: 163, 1953). There are no comparable studies, however, relating to bone or dentinal tissues. In view of the renewed interest in the histopathology of incipient dental caries and in the concept of a dental lymph suggested by the diffusion of diverse substances through dentin (C. F. Bodecker in A Survey of the Literature of Dental Caries, Washington, D. C., 1952, National Academy of Sciences National Research Council, Chapt. 5, p. 175), it seemed desirable to investigate the possible distribution of plasma albumin in intact dentin of the albino rat. Such an effort appeared feasible, despite the low concentration of aqueous extractable protein in dentin (Karshan, M., Weiner, R., and Stofsky, M.: J. D. Res. 14: 445, 1934) since the fluorescent-labeled antibody technic under ordinary conditions is capable of detecting as little as 100 peg. antigen per milliliter. (Coons, A. H.: Internat. Rev. Cytol. 5: 1, 1956.) Gamma globulin fractions of rabbit antisera to rat albumin* were prepared by the method of Dubert, Slizewicz, and Macheboeuf (Ann. Inst. Pasteur, Par.: 84: 370, 1953). Anti-rabbit -y globulin sheep globulin labeled with fluorescein isothiocyanatet was employed in the sensitive layer technic as reviewed by Coons (loc. cit.). Both globulin preparations were absorbed with mouse and bovine liver powder according to the method of Coons and Kaplan (J. Exper. Med. 91: 1, 1950). Fourteen frozen, undecalcified hemi-mandibular sections from Osborne-Mendel strain albino rats, 19 to 42 days of age and maintained on Purina Laboratory Chow, were prepared by the method of Steinman, Hewes, and Woods (J. D. Res. 38: 592, 1959). The sections were fixed according to the techi-ic of Vazquez and Dixon (J. Exper. Med. 104: 727, 1956). The staining and controls followed the protocol of White (Workshop on Fluorescent Antibody Techniques, U. S. Army Chemical Corps, Fort Detrick, Frederick, Md.). The sections were examined microscopically for fluorescence at 100x and 950x. Specific fluorescence referable to plasma albumin was readily demonstrable in the dentinal tubules for at least the proximal half of their course and in the pulpal margin of the dentinal matrix. Staining was more pronounced in specimens taken from the younger animals. The controls did not exhibit the characteristic fluorescence. Further studies of plasma protein distribution in dentin-particularly of the globulins should be of significance in assessing the dentinal tissue response to infectious agents.

The Pathogenic Role of a Rat Oral Streptococcus in Incipient Dental Caries

Recent studies on gnotobiotic rats have shown a causal relationship between an oral streptococcus and the pathogenesis of dental caries (R. J. Fitzgerald, personal communication). The streptococcus is a versatile micro-organism apparently capable of invading tissue, of elaborating biologically active cells, acids, enzymes, and toxins, and, like other organisms, of eliciting a delayed hypersensitive response. Considerable attention has been given to the significance of acidogenesis, with less consideration to the other microbial properties. It seemed desirable, therefore, to extend our definition of the relationship between this organism and the incipient carious lesion. Rat oral streptococcus strain FA-1 secured from Dr. Robert J. Fitzgerald, of the National Institute of Dental Research, was routinely cultivated on a yeast extract-peptone-dextrose broth for 24 hours at 370 C. Osborne-Mendel rats between 20 and 50 days of age and maintained on either Purina laboratory chow or the cariogenic diet previously reported (R. R. Steinman, C. G. Hewes, and R. W. Woods, J. D. Res., 38:592, 1959) were employed in this study. The animals were sacrificed and the jaws prepared by the method of Steinman et al. (loc. cit.) and fixed by the method of Vazquez and Dixon (J. Exptl. Med., 104:727, 1956). Incipient lesions were recognized by localized changes in ninhydrinreactive protein, in dehydrogenase activity, and in availability of sulfhydryl radicals (Steinman et al., loc. cit.; R. W. Woods, J. D. Res., 38:592, 1959; Steinman, R. R., J. D. Res., in press). Systemic inoculation of organisms.-Intracutaneous and intraperitoneal inoculation of forty-seven rats, 20-30 days of age, with 0.25 ml. of a washed-cell suspension had no observable systemic effect on the rats over a period of 20 days and did not alter the incidence of incipient carious lesions. Neither a systemic toxicity of the organisms nor their allergenicity appeared to influence the pathogenesis of the lesions. Fluorescence localization of organisms.-Sections from thirty-two rats, 20-50 days of age, were stained with rabbit anti-streptococcal globulin conjugated with fluorescein isothiocyanate by the method of Marshall, Eveland, and Smith (Proc. Soc. Exptl. Biol. Med., 98:898, 1958). In addition to routine controls, specificity of staining was established on suspensions of the homologous organisms, on fresh isolates from rat oral washings, and by the failure of staining of heterologous bacteria. The sections frequently exhibited fluorescence referable to the organisms in the debris of the grooves, rarely showed fluorescence in the superficial layers of the enamel at the bases of the grooves, and never in the underlying dentin. Penetration of these organisms did not discernibly occur in these early lesions. In vitro application of lysate.-In view of the fact that a lysate of this streptococcus has been shown to possess pronounced biological activity (J. D. Zwemer and R. R. Steinman, J. D. Res., in press), it seemed appropriate to test such a preparation on intact murine dentinal tissue. Lysate prepared by the method of Stetson (J. Exptl. Med., 104:921, 1956) was incubated with the sections for 2 hours, and then the sections were examined for dehydrogenase activity and sulfhydryl availability. Control observations were made on sections exposed to buffered saline and to the alkaline saline supernatant of homogenized glass beads. Sections from more than one hundred rats consistently indicated a specific, marked diminution in dehydrogenase activity and diminished availability of sulfhydryl radicals. These generalized histochemical changes were identical with those observed locally in the natural lesion. Efforts are now being made to characterize the streptococcal component(s) involved and the exact nature of the affected dental substrate.

The Endotoxic Activity of a Rat Oral Streptococcus

Until recently it was assumed that endotoxic activity was restricted to the Gram-negative bacteria. Now it is known that reactions induced by endotoxins, such as the delayed skin response, local and generalized Shwartzman phenomena, febrile response, and the potentiation of epinephrine, are also elicited by lysates of Group A beta hemolytic streptococci (C. A. Stetson, J. Exptl. Med., 104:921, 1956). In view of the fact that streptococcal species have been shown to produce dental caries in gnotobiotic rats (F. J. Orland, J. D. Res., 33:145, 1954; R. J. Fitzgerald, personal communication), it seemed desirable to establish the possible endotoxic activity of such an organism. Rat oral streptococcus strain FA-1 which induces dental caries in gnotobiotic rats was secured from Robert J. Fitzgerald, of the National Institute of Dental Research. The organism was maintained on microassay culture agar* containing added calcium carbonate and was cultivated on modified microinoculum broth for 48 hours at 370 C. Streptoccocal lysate was prepared and employed according to the method of Stetson (loc. cit.), using pyrogen-free distilled water and physiologic saline. E. coli (026:B6) lipopolysaccharide* and physiological saline (0.15 M NaCl) were employed for reference and control purposes. Twenty-eight normal albino rabbits weighing approximately 1.5 kg. were used as experimental animals. The animals were maintained on Wayne rabbit diet in quiet, thermostatically controlled quarters. Techniques for eliciting the delayed skin reaction, local and generalized Shwartzman, and the febrile responses followed those of Stetson (loc. cit.). The method of Thomas (J. Exptl. Med., 104:865, 1956) was employed for the demonstration of endotoxin potentiation of epinephrine. Each of the several local phenomena was typically observable as a 1-3-cm. skin lesion. Delayed local inflammatory reaction.-Intradermal injection of 0.4 ml. streptococcal lysate into each of five rabbits elicited characteristic erythema, induration, and edema, which was readily demonstrable at 24 hours and thus consistent with the observations of Stetson (loc. cit.). Saline-injected sites were non-reactive. Local Shwartzman.-Intravenous injection of 200 Pg. E. coli endotoxin at the height of the skin reactions in these same rabbits caused the development of severe hemorrhagic necrosis at the initially reactive sites. Generalized Shwartzman.-One of five rabbits prepared with intramuscular injections of cortisone acetate (35 mg/day) for 3 days developed cortical necrosis following provocation with 2.0 ml. streptococcal lysate intravenously. Febrile response.-Four animals injected intravenously with either 2.0 ml. lysate or a washed cell suspension and followed for 4 hours showed a significant elevation of body temperature when compared with four animals injected with pyrogen-free, physiologic saline. Epinephrine potentiation.-Five rabbits injected intravenously with 2.0 ml. lysate and then intradermally with 0.2 ml. physiologic saline at one site and 0.2 ml. epinephrine (1:1000) in physiologic saline at a second site uniformly developed severe hemorrhagic necrosis at the epinephrine site within 24 hours. Reactions were identical with those observed in an equal group of rabbits similarly tested with 200 jug. E. coli lipopolysaccharide.

Pediatrics in General Practice

For general practitioners, in whose practice is included a major proportion of the medical management of infants and children, and for the specializing pediatrician as well, Postgraduate Medicine presents this special regular department devoted to brief discussions by recognized authorities on their preferred methods of the treatment and management of diseases and problems of infancy and childhood.