Effectiveness of neuraminidase in experimental immunotherapy of two murine pulmonary carcinomas.

The effects of direct intratumoral inoculation with Vibrio cholerae neuraminidase and inoculation of tumor-bearing mice with tumor cells incubated with neuraminidase in vitro were studied in C57BL/6 X DBA/2 F1 mice bearing s.c.-transplanted, methylcholanthrene-induced pulmonary squamous cell or Lewis lung carcinomas. The growth of the squamous cell tumor was more greatly inhibited by both treatments than was the Lewis lung tumor. In the squamous cell tumor-bearing mice, both modes of neuraminidase treatment depressed tumor growth by approximately 80%. However, 20% of the mice in the group treated with the neuraminidase-incubated squamous cell vaccine and 10% of those treated intratumorally underwent total tumor regression and developed specific immunity to the squamous cell tumor. although the growth rate of the Lewis lung tumor was suppressed by both types of treatment, the direct intratumoral neuraminidase treatment group underwent a greater depression in tumor growth (73 versus 42%). A possible explanation of the different results of the two treatments in squamous cell and Lewis lung tumor systems may be based on tumor etiology and cellular composition.

Synthesis and secretion of IgA, IgM, and IgG by peripheral blood mononuclear cells in human disease states, by isolated human intestinal mononuclear cells, and by human bone marrow mononuclear cells from ribs.

We have examined the secretion of IgA, IgM, and IgG by isolated human intestinal MNC, human bone marrow MNC from rib specimens, and peripheral blood MNC from patients with CD, UC, SLE, and HSP. "Normal" control intestinal MNC exhibited high spontaneous secretion of IgA, whereas intestinal MNC from UC and CD patients exhibited only modest increases in IgA secretion. Peripheral blood MNC from patients with CD, UC, SLE, and HSP exhibited markedly elevated spontaneous secretion of immunoglobulins in general and IgA in particular. Pure human bone marrow MNC exhibited high spontaneous secretion of IgA with modest amounts of IgG and normal IgM being secreted. The addition of PWM to cultures in which high spontaneous synthesis and secretion of immunoglobulins was seen, resulted in no further enhancement, and in some instances suppression, of antibody secretion. In patients with autoimmune disease, there appeared to be dual immunoregulatory defects, one involving a lack of normal T-suppressor cell functional capabilities for spontaneous antibody synthesis, and the other the presence of PWM activable T-suppressor cells. In human bone marrow, we have identified MNC that secrete suppressor factors in the presence of PWM and that are capable of inhibiting antibody synthesis and secretion. Column separation using Sephacryl S-300 revealed that the IgA secreted by "normal" control intestinal MNC is predominantly dimeric, whereas the IgA secreted by human bone marrow MNC is predominantly monomeric. Furthermore, mucosal MNC from patients with CD and uninvolved intestine from patients with UC exhibited patterns similar to control intestinal MNC, being predominantly dimeric IgA with some monomeric IgA secreted. By contrast, intestinal MNC from patients with UC had a decreased proportion of dimeric IgA and increased proportion of monomeric IgA, thus indicating that IgA precursor B-cells may have migrated into the intestine from extraintestinal sites, or that the normal dimeric IgA-secreting cells in the intestine had begun secreting increased proportion of monomeric IgA as well. These studies indicate that homing patterns and/or immunoregulation of IgA-secreting cells are altered in human intestine, bone marrow, and autoimmune disease states.

Antiserum-induced growth of axons across lesions of the adult rat brain.

Growth of axons across lesions of the adult rat brain occurred when the lesions were treated with a heterologous antiserum developed against lesioned areas of the brain. Rabbits were immunized with blocked dissected damaged rat brain plus adjuvant. The antiserum was prepared by ammonium sulfate precipitation of the immune rabbit sera followed by pepsin digestion to prevent complement-mediated damage to the recipient rats. Rats treated with the antiserum had dense cellular bridges crossing the brain lesions which contained axons. The axons within the dense cellular bridges were newly formed, since they were observed to pass through the center of paper rings which were implanted into the lesion. Individual axons were traced from one side of the lesion, through a dense cellular bridge, and into the tissue on the opposite side of the lesion. Rats lesioned in a similar manner, but treated with either phosphate-buffered saline or normal rabbit serum displayed no such growth. In addition, the limited axonal growth observed was enhanced by increasing the concentration of the antiserum administered. Thus, the antiserum induced the formation of dense cellular bridges and the growth of axons across lesions of the mammalian central nervous system.

Human bone marrow-derived IgA is produced by IgA-committed B cells in vitro.

Although human bone marrow has been implicated in the production of serum immunoglobulins, little information is available concerning the kinetics of antibody production (primary- or secondary-type humoral response) or the cells that are responsible for antibody production in human bone marrow. In this study, the kinetics of and cells responsible for antibody production in the bone marrow were investigated. Previous studies have demonstrated that human bone marrow mononuclear cells secrete a significant amount of IgA in vitro. This finding led to the focus of the present investigation on bone marrow IgA production. The results reported here demonstrate that IgA was synthesized de novo in cultures of bone marrow mononuclear cells; its peak concentration in the culture supernatants preceded that of IgM; its production was totally inhibited by the addition of microgram quantities of anti-alpha-chain antiserum, while milligram quantities of anti-mu-chain antiserum were required for even partial inhibition of IgA production; and the culture of isolated IgA-bearing cells resulted in a greater than 13-fold increase in IgA concentration in the culture supernatants as compared with those from unseparated bone marrow mononuclear cells. From this study, it was concluded that bone marrow produces IgA as a secondary or anamnestic response to some undetermined stimulus and that IgA-committed B cells residing in, although probably not stimulated in, the bone marrow compartment were responsible for the IgA synthesis and secretion in vitro.

Marked in vitro spontaneous secretion of IgA by human rib bone marrow mononuclear cells.

Mononuclear cells (MNC) isolated from bone marrow curettaged from human ribs spontaneously produced significant amounts of Iga in 14-day in vitro culture. When culture supernatants were tested for each immunoglobulin isotype by radioimmunoassay at days 7, 14, 21, and 28, the peak of spontaneous IgA production was reached by day 14. Culturing marrow MNC in the presence of the polyclonal activator pokeweed mitogen (PWM) ablated this spontaneous IgA activity, and did not stimulate the production of IgG or IgM. In contrast, peripheral blood MNC produced significant amounts of all three immunoglobulin isotypes studied in the presence of PWM, but minimal immunoglobulin spontaneously. PWM stimulated both marrow and peripheral blood MNC to proliferate as demonstrated by increased DNA synthesis. In view of the opposite effects of PWM on immunoglobulin production by peripheral blood and bone marrow MNC, PWM may be stimulating a different functional or developmental subpopulation of cells in each case. We conclude that the bone marrow is a major site of IgA production in the human, that cells within the bone marrow have a blastogenic response to PWM in vitro, and that spontaneous production of IgA by human marrow MNC is inhibited by PWM.

Murine bone marrow IgA responses to orally administered sheep erythrocytes.

Specific immunization protocols have been established for the induction of murine bone marrow IgA responses to the T cell-dependent (TD) antigen sheep red blood cells (SRBC). Systemic immunization, either i.p. or i.v., followed by a second injection, induced splenic IgM and IgG responses and a bone marrow IgM response. No significant IgA responses were observed in either lymphoid tissue compartment. Oral immunization with SRBC by gastric intubation for 2 days, followed 1 wk later by an i.p. injection of SRBC resulted in a splenic IgA plaque-forming cell (PFC) response, but did not elicit a bone marrow IgA response. Repeated daily gastric intubation of SRBC to C3H/HeN and C3H/HeJ mice led to the previously reported pattern of systemic unresponsiveness in C3H/HeN mice and good anamnestic type IgM, IgG, and IgA splenic anti-SRBC PFC responses in the C3H/HeJ strain upon parenteral challenge. Oral administration of SRBC for 14 days to C3H/HeN mice, followed by systemic SRBC challenge, resulted in diminished splenic PFC responses of all isotypes, whereas gastric intubation of SRBC for 28 days led to complete systemic unresponsiveness to antigen in C3H/HeN mice. Interestingly, the repeated oral administration of SRBC resulted in significant bone marrow IgA PFC responses upon i.p. challenge in both C3H/HeN and C3H/HeJ mouse strains. The bone marrow IgA responses were clearly dependent upon chronic oral exposure to SRBC, because gastric intubation with SRBC for 2 consecutive days/wk for 10 wk also induced bone marrow and splenic IgA anti-SRBC PFC responses in C3H/HeN mice. These results suggest that memory B cells reside in the bone marrow of orally immunized mice and can yield anamnestic-type responses to challenge with the inducing antigen. The memory cells may arise in the Peyer's patches of the gut and migrate to the bone marrow. The possibility that the bone marrow is a component of the common mucosal immune system in mammals is suggested by this study.

The effect of serum on human marrow mononuclear cell proliferation and maturation.

The effect of different serum sources on the growth of human bone marrow mononuclear cells in liquid culture was investigated. Newborn calf serum failed to support growth in liquid cultures whether or not exogenous colony-stimulating factor was present. Neither adherent nor nonadherent cells proliferated in medium supplemented with horse serum. Fetal calf serum allowed proliferation of the nonadherent cell population, but only in the presence of colony-stimulating factor. However, no growth of adherent cells was observed in these cultures. Both the nonadherent and adherent populations grew well in the presence of pooled human sera. While growth of the nonadherent population was minimal in the absence of colony-stimulating factor, the adherent population appeared to increase to a greater extent when the factor was absent. The positive effect of the human serum was shown to be dose dependent in that as the proportion of serum was increased in the medium, the number of cells recovered from the cultures increased, regardless of the presence or absence of exogenous colony-stimulating factor.

Immunochemical localization of amiloride-sensitive sodium channels in sodium-transporting epithelia.

The localization of amiloride-sensitive Na+ channels in Na+-transporting epithelia was examined using antibodies made against amiloride-binding Na+ channel protein purified from bovine kidney. The distribution of the channel protein was determined in thick frozen sections at the light-microscopic level using indirect immunofluorescence, and at the electron-microscopic level using immunogold labelling. In the cells of both the intact bovine collecting tubule and A6 confluent monolayers, only the luminal or apical-facing surface membranes showed staining. Sodium channel protein was characteristically localized on microvillar domains of the apical plasma membrane. Little or no basolateral membrane staining was evident. Channel protein was also absent from subapical vesicles and tight junctions, and was not found in bovine renal proximal tubules, cultured human secretory sweat coils, non-epithelial Chinese hamster ovary (CHO) cells or human skin fibroblasts. Trypsinization of intact A6 monolayers prior to cell fixation abolished specific staining with antibody. Pretreatment with amiloride protected against this loss of staining. Thus, our probes are specific for amiloride-binding Na+ channel protein, and this channel protein is largely or completely confined to the apical membrane of Na+-transporting epithelia. The level and distribution of specific immunostaining in A6 cells was unchanged by aldosterone treatment, although channel activity, as measured by short-circuit current, increased threefold. This result demonstrates that Na+ channel protein is ever present at the cell surface and exists in both an active and an inactive form. We find no evidence that stimulation of Na+ uptake by aldosterone involves recruitment of new channels from a cytoplasmic pool.

Suppression of in-vitro antibody production by pokeweed mitogen-stimulated human bone-marrow mononuclear cells. Evidence for a soluble suppressor substance.

The spontaneous synthesis and secretion of immunoglobulin by human bone-marrow mononuclear cells (MNC) in vitro, as well as its suppression by the addition of pokeweed mitogen (PWM), were previously reported by this laboratory. In the present study we demonstrate that this suppression is mediated by a soluble substance elaborated by marrow MNC stimulated with PWM. Marrow MNC were pulsed for 1 hr with PWM, washed and recultured for 7 days in media without PWM. The culture supernatants were collected by centrifugation and filter sterilized before addition to fresh marrow MNC in the in vitro antibody synthesis assay. The 14-day assay culture supernatants were then subjected to a solid phase radioimmunoassay to determine the immunoglobulin content. The suppressor substance was non-specific as to immunoglobulin isotype and was not genetically restricted. Suppressor activity was diminished by heating the supernatants at 56 degrees for 1 hr. The activity could be elaborated by cells subjected to 1000 R or 2000 R before or after 1-hr incubation with PWM. While the addition of PWM anytime during the culture period would suppress IgA production at the level produced up to that time, the suppressor substance only suppressed IgA production when added during the first 4 days of culture. The addition of indomethacin had no effect on the suppressor activity indicating that the activity was not mediated by prostaglandin. Including human fibroblast interferon or hydrocortisone in the assay cultures had no effect on IgA production or its suppression by PWM. We concluded that the lectin-induced suppression was mediated by a marrow-derived suppressor substance (MDSS).

Subpopulations of B lymphocytes in germinal centers.

With two new monoclonal antibodies and flow cytometry, we defined three subpopulations among B cells expressing binding sites for peanut agglutinin (i.e., B cells of the germinal center). On monoclonal antibody (5B5) binds globotriaosyl ceramide. The B lymphocytes binding 5B5 have binding sites for peanut agglutinin on the surface and express only small amounts of sIgD and sIgM. When tested against a panel of B cell lines, only Burkitt's lymphoma cells were 5B5+. Moreover, the 5B5+ cells have larger average sizes and a large fraction of proliferating cells. The other monoclonal antibody (HK23) binds a 90,000 protein. Lymphocytes binding HK23 are 5B5- and include T cells and a subpopulation of B cells. In contrast to 5B5+ cells, the HK23+ and peanut agglutinin positive B cells express a large amount of sIgM. These two subpopulations of germinal centers are distinct from the germinal center B cell subpopulation expressing the CD23 (Blast-2) antigen. The CD23+ B cells are 5B5- and express an intermediate level of HK23 antigen. In addition, CD23+ B cells are highly variable in number, whereas the proportions of HK23+ and 5B5+ cells are relatively stable.