Modification of in vitro and in vivo BCG cell wall-induced immunosuppression by treatment with chemotherapeutic agents or indomethacin.

The in vitro inhibition of spleen cell blastogenesis response and the in vivo enhancement of tumor growth are phenomena associated with BCG cell wall (BCGcw) immunization. What effect treatment with chemotherapeutic agents and the prostaglandin inhibitor indomethacin would have on the in vitro and in vivo responses to BCGcw immunization was evaluated. In vitro blastogenesis studies showed that chemotherapy pretreatment prior to immunization with BCGcw resulted in a restoration of the spleen cell blastogenesis response. In blastogenesis addback studies, where BCGcw-induced irradiated splenic suppressor cells were admixed with normal cells, less inhibition of blastogenesis occurred when spleen cells were obtained from rats that had received the combined treatment of chemotherapy and BCGcw immunization versus only BCGcw immunization. The cocultivation of spleen cells from BCGcw-immunized rats with indomethacin resulted in a 30-40% restoration of the blastogenesis response. In vivo studies showed that BCGcw-mediated enhancement of intramuscular tumor growth of the 3924a ACI rat tumor could be abrogated by either pretreatment with busulfan or mitomycin or by the feeding of indomethacin.

The inhibition of fatal graft-versus-host disease by immunization of donor or host with bacillus Calmette-Guérin cell walls.

F1 mice receiving sublethal whole-body x-irradiation (300 rads) or treatment with cyclophosphamide prior to the i.p. inoculation of parental spleen cells developed fatal graft-versus-host disease (GVHD). A greater survival rate was obtained when the inoculated parenteral spleen cells were obtained from BCGcw-preimmunized donors. The immunization of the F1 host with bacillus Calmette-Guerin cell walls (BCGcw) also increased host survival. The combined treatment of preimmunizing the host with BCGcw and of using spleen cells from BCGcw-immunized parental donors to initiate the GVHD resulted in producing the least severe GVHD and the greatest overall survival. The systemic transfer of x-irradiated spleen cells from BCGcw-immunized parental mice inhibited the fatal GVHD induced by the inoculation of normal parental spleen cells. These studies show that BCGcw immunization of the host or obtaining parental spleen cells from BCGcw-immunized animals resulted in improving the overall survival rate in graft-versus-host disease. BCGcw immunization induces suppressor cells and the decrease of graft-versus-host disease that was observed was most likely due to the induction of suppressor cells.

Comparative studies on tumor and adjuvant (BCGcw)-induced nonspecific suppressor cells in rats.

Comparative studies on tumor and adjuvant-induced depression of in vitro mitogen responses were carried out using spleen cells obtained from syngeneic tumor bearing (TB) ACI rats or from rats which had been immunized with BCG cell walls attached to oil droplets (BCGcw). These in vitro studies demonstrated that: 1) the spleen cells from TB rats (TB-spleen cells) showed strongly depressed mitogen responses to concanavalin-A (Con-A), phytohemagglutinin-P (PHA-P) and lipopolysaccharide (LPS), 2) the mitogen response of lymph node cells from TB rats was slightly depressed, 3) the removal of plastic or nylon-wool adherent cells or phagocytic cells from TB-spleen cells resulted in a restoration of the mitogen response, 4) the Con-A response of normal spleen cells could be suppressed by the addition of TB-whole spleen cells, 5) the suppressor cell activity was not abrogated by the in vitro treatment with x-irradiation (2000 rads), 6) carbonyl-iron treated TB-spleen cells showed a normal level of mitogen response, and on addback to normal spleen cells no suppressive activity was detected in them. Similar results were observed when spleen cells were obtained from BCGcw immunized rats. These results suggest that in ACI rats tumor-induced nonspecific suppressor cells detected by in vitro assay are the same cell populations as BCGcw-induced nonspecific suppressor cells.

Negative and positive immunobiological responses in mice pretreated with Bacillus Calmette-Guérin cell wall.

Spleen cells from C57BL/6 mice injected i.p. with Bacillus Calmette-Guérin cell walls (BCGcw) showed strongly depressed response to the T-cell mitogen phytohemagglutinin-P in vitro. Mitogen reactivity of normal spleen cells could be suppressed by the addition of spleen cells from BCGcw-treated mice. The suppressor cells mediating this effect appeared to belong to the plastic-adherent, radioresistant, and non-T-cell populations, maybe macrophages. Spleen cells from mice which had been passively transferred i.p. with the adherent cells from BCGcw-treated mice also showed the depressed mitogen response in vitro. Depressed T-cell reactivity of spleen cells obtained from animals immunized with BCGcw on a per-cell basis was also demonstrated in vivo: graft versus host reactivity of spleen cells obtained from animals immunized with BCGcw was depressed as compared to normal spleen cells. At times when strong suppressor cell activity could be detected in BCGcw-treated mice, activity of alloimmune cytotoxic lymphocytes generated in vivo by immunizing with X-irradiated allogeneic MH-134 tumor cells was weaker in BCGcw-pretreated mice than in untreated control groups (detected by means of 51Cr release assay). Furthermore, accelerated development of s.c. inoculated syngeneic B-16 melanoma cells was observed in BCGcw-pretreated mice. On the other hand, stronger resistance to i.v. inoculated B-16 tumor cells was observed in BCGcw-pretreated mice. BCGcw-treated mice responded normally to i.p. immunization with 2 X 10(8) sheep erythrocytes. Negative and positive immunobiological responses were observed in C57BL/6 mice pretreated with BCGcw.

Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1.

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.

Glucocorticoid and antibiotic effects on hepatic microcirculation and associated host responses in lethal gram-negative bacteremia.

Liver changes and associated host responses were evaluated in four groups of male rats, weighing 300 +/- 20 gm., which received intravenous injection of 2.2 times 10(9) live Escherichia coli. This bolus was given either without additional treatment (group A) or prior to the following regimens: intramuscular injection of gentamicin sulfate, 5 mg. per kg. (group B); intravenous injection of methylprednisolone sodium succinate, 40 mg. per kg. (group C); and intramuscular injection of gentamicin immediately after methylprednisolone sodium succinate treatment (group D). Rats given injections of saline or methylprednisolone sodium succinate served as controls. Survival rates at 10 and 20 hours were 25 per cent and 4 per cent for group A; 44 per cent and 28 per cent for group B; 94 per cent and 70 per cent for group C; 98 per cent and 98 per cent for group D, respectively. In rats of groups A and B, killed at 1, 2, 4, and 6 hours, progressive liver changes included intravascular sequestration of rapidly degranulating leukocytes, fibrinous deposits, and platelet aggregates in sinusoids as well as in spaces of Disse adjacent to subendothelial collagen, and extensive Kupffer cell disruption in association with severe midzonal necrosis. These alterations were accompanied by progressive hypoglycemia and elevations of serum enzymes, glutamic pyruvic transaminase, lactate dehydrogenase, and glutamic oxaloacetic transaminase. Hematologic studies revealed that E. coli bacteremia results in rapid leukopenia and disseminated intravascular coagulation primarily due to activation of the intrinsic coagulation pathway. All above reactions were delayed and markedly reduced in rats treated with methylprednisolone sodium succinate. The results indicate that antibiotic treatment of lethal, Gram-negative bacteremia is effective only in conjunction with early steroid treatment. The protective effects of glucocorticoids on the liver microcirculation and polymorphonuclear leukocytes appear to play a basic role in preventing the early development of disseminated intravascular coagulation, hepatocellular necrosis, and associated major host responses, thereby attenuating lethality of gram-negative septic shock.

Effect of deworming medication on the microbial flora of the upper gastrointestinal tract of dogs.

Control cultures were taken at laporatomy of the stomach or stomach remnant, jejunum, and ileum of 26 adult dogs, 8 of which had had gastric surgery 1 yr earlier. Two wk after the control cultures were taken, 17 of the 26 dogs, including all 8 which had had prior surgery, were treated with anthelmintics, niclosamide, dichlorophene, methylbenzene, and arecoline hydrobromide. Two wk after the anthelmintic treatment, or 4 wk after the control cultures were taken, the 26 dogs were recultured. Samples of microbial flora were obtained by direct needle aspiration employing anaerobic precautions. These samples were processed both quantitatively and qualitatively using both anaerobic and aerobic technics. The specific sites cultured were the lower stomach or stomach remnant, proximal jejunum 15 cm distal to the ligament of Treitz, and distal ileum 45 cm proximal to the ileocecal valve. Bacteria isolated were predominately facultative aerobes. No fungi were isolated. Cultivation of spirochetes was not attempted. The results showed that there was no significant qualitative or quantitative alteration of microbial flora caused by the anthelmintic treatment.

Murine cytomegalovirus glomerulonephritis: clinical and ultrastructural observations.

This investigation was initiated to study and correlate the clinical and ultrastructural aspects of glomerulonephritis induced in the laboratory mouse by the intraperitoneal injection of a sublethal dose of murine cytomegalovirus. An attempt was made to ascertain the pathogenesis of the glomerular changes and the resultant viremia. Murine cytomegalovirus infection caused an acute transient glomerulonephritis in young female mice of the HA/ICR strain. Mice that survived a sublethal inoculation of homogenized infected gland developed transient proteinuria and excreted tubular casts. The murine cytomegalovirus infection resulted in a glomerular lesion that was selective for the mesangial cell. After entering the mesangial cell by phagocytosis the virus replicated in the nucleus and was excreted into the channel of the mesangial matrix, with extension toward the periphery of the capillary loop and adjacent to the urinary space. Virus particles were rarely found in the glomerulus after the 5th day of infection and chronic renal disease was not observed.