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The subcutaneous route (s.c.) was used to study the adjuvant effect of Bordetella pertussis vaccine (PV) on the primary antibody response to sheep erythrocytes. The reasons for using the s.c. route are discussed. PV, besides enhancing the hemagglutinin response, also markedly increased the number of plaqueforming cells in the draining lymph nodes. A heated preparation of PV was tested and found to possess significant adjuvant activity. Interestingly, the enhancement occurred in the absence of marked enlargement of the lymph nodes, which was characteristic of the unheated preparation. In addition, a crude solubilized cell-free preparation of PV was tested and also found to possess significant adjuvant activity. The activity was only partially abolished by heat.Hence, it was concluded that both heat-labile as well as heat-stable factors contributed to the adjuvanticity of PV. The studies also support the view that the draining lymph nodes represent a principal locus of action of PV and that the s.c. route of administration of adjuvant and antigen provides a suitable model for studying and assaying the adjuvanticity of PV.

Granuloma formation has been the concern of many investigators at both the research and clinical levels. The procedures used to study granulomatous inflammation have included morphological observation ( 1 ), analysis of cell migration patterns (2-4), and measurement of granuloma size in terms of granuloma diameter ( S ) , volume ( 6 ) , and weight ( 7 ). These methods, however, lack the capacity to quantitate the actual number of each cell type involved in the formation of a granuloma.Our laboratory has been particularly concerned with quantitation procedures for the study of the inflammatory exudate in the peritoneal cavity (8). In the course of these studies, it was noted that granulomatous tissue was subsequegtly formed at the site of inflammation (9, lo). Our investigations were then extended to determine if it would be possible to develop a technique for quantitating the cells in the granuloma. This study describes a procedure for dispersing experimentally induced granulomas into their cellular components. Also presented are the results obtained when this procedure was applied at different stages of granuloma formation following the injection of antigenic and nonantigenic substances.ulomas. In this investigation, 3 types of granulomas were induced by subcutaneous injections into the inguinal region of adult female BDFl hybrid mice. Type I (primary granuloma) consisted of granulomas formed in response to a priming injection of 0.4 ml of tetanus toxoid adsorbed onto aluminum phosphate (APTT) (Parke Davis). Type I1 (secondary granuloma) consisted of granulomas formed in response to a challenging injection of 0.4 ml of APTT in mice which had been primed subcutaneously in the dorsal neck 5 weeks previously with 0.2 ml of APTT mixed with 0.2 ml of pertussis vaccine (PV) (Eli Lilly) adjuvant diluted 1 : 10 with saline. Type I11 (alum granuloma) consisted of granulomas formed in response to an injection of 0.4 ml of a nonantigenic substance, aluminum phosphate (AP), ( 5 mg/ml of saline) into mice which had been primed with PV-APTT as above. In order to facilitate location of the granulomas, activated sterile carbon (C) (Merck and Co.) was mixed with AP, APTT and PV-APTT solutions in a concentration of 2 mg/ml.Processing of granulomas. Mice were killed by cervical dislocation, dipped quickly into a solution of pHisoHex (Winthrop Laboratory, New York), and the skin was peeled back to reveal the subcutaneous granulomas.Each granuloma was freed of any adherent connective tissue and then subjected to dispersion and digestion procedures in a siliconized tube containing 1 ml of a cold, freshly prepared solution of collagenase.Collagenase, from Clostridium histo2yticum (General Biochemical Corp.) was prepared at a concentration of 4 mg of collagenase/ml of Hanks' balanced salt solution (pH 7.1-7.2). After 1-3 hr of incubation at 37", the stroma dissolved; and the granuloma fell 1090

Intravenous injection into mice of phase I Bordetella pertussis culture supernatants produces a marked lymphocytosis. The evolution of lymphocytosis was correlated with histopathological alterations in the lymphoid organs. The early phase, 17 hr to day 3, was associated with massive depletion of both lymphocytes from the white pulp of the spleen and of cortical thymocytes. There was a corresponding profound decrease of splenic and thymic weights. The later phase was associated with a marked decrease in lymphocytes of lymph nodes while concomitantly the spleen and thymus appeared to be repopulating. Both thymic-dependent and thymic-independent lymphocyte populations were mobilized from spleen and lymph nodes. There was a striking diminution in the number of lymphocytes contained within the walls of postcapillary venules (PCVS) of all lymph nodes between 2 and 7 days. The data suggest that initially the lymphocytosis is due predominantly to release of splenic lymphocytes and to a lesser extent of thymocytes into the circulation. Subsequently, the lymphocytosis is sustained by the depopulation of the lymph nodes. Lymph node depopulation is maintained by the failure of circulating lymphocytes to emigrate from the blood through the PCVS.