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The activity of Nocardia water-soluble mitogen (NWSM) and LPS were compared in several experimental systems, since both compounds are B-cell mitogens and polyclonal activators in vitro. The results reported here demonstrated that NWSM like LPS also has a strong adjuvant activity in vivo if administered in saline with a strong antigen (heterologous red blood cells) or even with a weak immunogen such as theta alloantigen. However, in contrast to LPS, NWSM administered to mice failed to induce in vivo proliferation of lymphocytes, polyclonal activation and PFC against syngeneic bromelain-treated erythrocytes and thymocytes. It is possible therefore, that different mechanisms may be responsible for adjuvant activity of NWSM and LPS.

The in vitro synthesis of allotypes of b4/b5 offspring obtained from b4/b4 mothers immunized against paternal allotype b5/b5 was studied in comparison to similar offspring that had escaped from suppression and normal heterozygous b4/b5 rabbits. Nocardia water-soluble mitogen-a rabbit B-cell mitogen which is known to induce the differentiation of small lymphocytes into plasma cells and polyclonal activation of Ig, was able to break in vitro the allotypic suppression induced in vivo.

Introduction of the CBA/N X-linked gene into C3H mice has resulted in the establishment of a new strain of mice that has profound immunologic defects. B cells from these mice show significantly impaired in vitro immune responses to the T cell-independent type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA) as well as markedly reduced proliferative responses to a number of B cell mitogens when compared with the responses of the parental control mice. The in vivo response of such mice to TNP-BA is, however, comparable to that of CBA/N mice. Furthermore, B cells from C3.CBA/N mice are unresponsive to the plaque-forming cell enhancing effects induced by EL4-derived supernatant in the presence of TNP-BA, unlike B cells obtained from CBA/N or C3H/Hen mice whose responsiveness to TNP-BA can be significantly enhanced in the presence of EL4-derived supernatant. The model we have presented to best explain these results suggests that B cells from C3.CBA/N mice can be stimulated only under conditions in which they can interact with carrier-specific T cell help and not under conditions where factor-dependent responses are dominant.

Nude mice lymphocytes were stimulated by heat-killed E.coli bacterial cells as well as by various components of their cell walls such as lipopolysaccharide, peptidoglycan and lipoprotein. Only bacterial cells, peptidoglycan and lipoprotein induce a high stimulation of C3H/HeJ mouse and rabbit lymphocytes. These data show the resemblance of mutant C3H/HeJ mice to rabbits with regard to blast response induced by various mitogenic components of E.Coli cell walls.

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.

A water-soluble extract of Nocardia markedly increased in vitro [(3)H]thymidine incorporation by mouse spleen lymphocytes. The blastogenic activity of the extract and lipopolysaccharide was studied comparatively on various mouse lymphocyte subpopulations. The data obtained by [(3)H]thymidine incorporation and by electron microscopy have demonstrated that this preparation stimulates selectively mouse bone-marrow-derived cortisone-sensitive lymphocytes. This stimulation is related neither to a natural infection of mice with Nocardia organisms nor to the presence in Nocardia water-soluble mitogen of a lipopolysaccharide contaminant or of a lipopolysaccharide-like material.

Delipidated lysozyme digests of Nocardia opaca, N. corallina, and N. rubra have been fractionated by Sephadex filtration. The mitogenic and adjuvant activities of the fractions thus obtained have been investigated. All fractions are mitogenic except the last fraction of N.rubra, but the N. opaca products induce a stronger stimulation of mouse spleen lymphocytes than the corresponding fractions of the two other species. The activity of the first Sephadex fractions of each strain has been compared to other mitogens (concanavalin A, lipopolysaccharide). All fractions are adjuvant, although one of them, the last Sephadex fraction of N. rubra, does not contain peptidoglycan; its activity must thus be attributed to another kind of molecule. Fractionation of the first Sephadex fraction of N. opaca by centrifugation in glacial acetic acid led to a separation of adjuvant and mitogenic activities.

Two purified peptides extracted from E. coli or B. megaterium strongly stimulated the spleen lymphocytes of rabbits and of normal or nude mice. Both preparations can substitute for Mycobacteria in Freund's complete adjuvant. The peptidoglycan extracted from M. lysodeikticus by a similar procedure which lacks adjuvant activity, did not induce blast transformation. However, the monomere of the E. coli peptidoglycan was devoid of mitogenicity although it has also a marked adjuvant activity.

Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.

The B-cell mitogens lipopolysaccharide (LPS), Nocardia water-soluble mitogen (NWSM), and dextran sulfate (DxS) react with different subpopulations of B lymphocytes. Selective in vitro killing of cells responding to either LPS or NWSM has little effect on the in vitro response to the other mitogen, although the response to DxS is reduced in both cases. If, after selective in vitro killing, cells are injected into irradiated mice for 2-3 wk before measuring their in vitro mitogen responses, the same specificity pattern is seen. Thus, one is dealing with different B-cell subpopulations rather than different stages of maturation of a single population. Treatment with various alloantisera and complement before measuring the mitogen response to LPS and NWSM shows that (a) whereas all LPS response cells carry surface Ig, a subpopulation of NWSM responsive cells does not; (b) both LPS- and NWSM-responsive cells carry I-A antigens but might not I-E or I-J antigens; (c) all LPS-responsive cells carry I-C antigens, whereas approximately 25% of NWSM responsive cells do not: (d) there is a subpopulation of NWSM-responsive cells carrying neither surface Ig nor I-C antigens and resistant to anti-theta treatment.

Two methods have been devised to isolate mitogens from Nocardia cells. (1) Lysozyme treatment of delipidated cells followed by fractionation of the soluble extract leads to a mitogen devoid of cell wall constituents. (2) Cell disruption followed by differential centrifugations shows that two mitogenic structures are present: the cell wall and fragments of the cytoplasmic membrane. The active component of the cell wall is the peptidoglycan wherefrom soluble mitogenic fractions have been obtained using bacteriolytic enzymes other than lysozyme. An intact glycan moiety seems necessary for mitogenic activity.

Fractionation of cells of Nocardia rubra and Nocardia opaca led to the separation of the cell wall and a "cytoplasmic" fraction. Both fractions were mitogenic for the splenocytes of AKR and nude mice and of rabbits. The peptidoglycan was the active part of the cell wall fraction. The products solubilized by the action of Streptomyces albus G peptidases on the peptidoglycan of N. rubra were mitogenic, but the products solubilized by lysoyme were not. Tentative structures are proposed for these fractions. The most active part of the cytoplasmic fractions could be sedimented by centrifugation and seemed to be related to the cytoplasmic membrane.

Nocardia water soluble mitogen (NWSM), a mitogen specific for mouse and rabbit B cells, activates blood, spleen, and tonsil lymphocytes from all the humans tested. Evidence is presented that it acts selectively on human B lymphocytes. B-enriched cells were recovered from anti-Ig-coated Sephadex columns or after elimination of E rosette-forming cells: they were able to incorporate tritiated thymidine when they were cultivated in the presence of NWSM whereas they were not activated by PWM. Conversely T-enriched suspensions did not respond to NWSM although they were able to be stimulated by PHA and PWM. Moreover, the response to NWSM remained unchanged after T cells had been killed by anti-HTLA serum and complement and disappeared after B cells had been killed by anti-HBLMA serum. After stimulation by NWSM, the number of direct plaque-forming cells to HRBC, LPS, and TNP was significantly increased, showing a polyclonal activation of human B lymphocytes. Since no evidence of T cell help for B cell activation has been found, NWSM thus appears to be a T-independent human B lymphocyte mitogen.