Antitoxic immunity against the so-called lung toxin produced by Escherichia coli.

Cross neutralization test with antisera to crude haemolysins produced by some Escherichia coli strains indicated that there were no antigenic differences among the haemolysins tested. These crude preparates showed definite cytotoxicity which could also be cross neutralized by "antihaemolysin" sera. Neutralization experiments were performed in mouse lung test with homologous and heterologous anti-haemolysin sera, and with O and OK sera to the wild type strain and its toxic R mutant. The results showed that the immunity in the mouse lung model is antitoxic and antibacterial.     

Cell-mediated immunity to viruses measured by the indirect agarose technique of leukocyte migration inhibition

The indirect agarose technique of leukocyte migration inhibition has been used to measure the response of human peripheral blood lymphocytes to several viruses. Using commercially available viral antigens, the indirect assay was found to be more sensitive than the direct agarose technique. Supernatants from cultures of sensitive lymphocytes with virus contained a nondialysable factor which inhibited the migration of polymorphonuclear leukocytes (PMN). Under strict conditions of assay, whereby all culture supernatants were tested together on the same PMN preparation, the degree of migration inhibition obtained in response to mumps virus correlated well with the size of the skin test reaction to mumps. A similar relationship was shown for PPD. A good correlation existed also between the degree of migration inhibition and the lymphocyte transformation response for each of these two antigens.     

Cellular immunity to Hodgkin's splenic tissue measured by leucocyte migration inhibition.

Patients with lymphoreticular malignancy were shown by a leucocyte migration inhibition technique to have cellular immunity to Hodgkin''s splenic tissue. Migration was significantly inhibited in 31 out of 55 patients with Hodgkin''s lymphoma and 19 out of 39 patients with other types of lymphoma. Inhibition was also shown in only three out of 29 patients with other malignancy, one out of 23 normal volunteers, and one out of 25 patients with non-malignant disease. The splenic factor that inhibits leucocyte migration, which has yet to be isolated and identified, may be a helpful diagnostic tool in patients with suspected lymphoma.     

Antioxidant-mediated inhibition of the heat shock response leads to apoptosis

We examined the hypothesis that reactive oxygen species (ROS) contribute to the induction of heat shock proteins (hsps) during stress response. Exposure of HL-60 human myelocytic cells to 42 degrees C induced both hsp72 and hsp27. In the presence of the antioxidant molecules pyrrolidine dithiocarbamate or 1,10-phenanthroline induction of hsp72 and 27 was significantly decreased, while N-acetyl-L-cysteine caused a slight reduction. Prevention of hsp induction was associated with heat sensitization and increased caspase activity, indicating that the cells were undergoing apoptosis. These data suggest that ROS contribute to the induction of hsps and furthermore, that hsp induction and apoptosis are mutually exclusive events within the same cell.     

Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein

Colicins are toxins secreted by Escherichia coli in order to kill their competitors. Colicin D is a 75 kDa protein that consists of a translocation domain, a receptor-binding domain and a cytotoxic domain, which specifically cleaves the anticodon loop of all four tRNA(Arg) isoacceptors, thereby inactivating protein synthesis and leading to cell death. Here we report the 2.0 A resolution crystal structure of the complex between the toxic domain and its immunity protein ImmD. Neither component shows structural homology to known RNases or their inhibitors. In contrast to other characterized colicin nuclease-Imm complexes, the colicin D active site pocket is completely blocked by ImmD, which, by bringing a negatively charged cluster in opposition to a positively charged cluster on the surface of colicin D, appears to mimic the tRNA substrate backbone. Site-directed mutations affecting either the catalytic domain or the ImmD protein have led to the identification of the residues vital for catalytic activity and for the tight colicin D/ImmD interaction that inhibits colicin D toxicity and tRNase catalytic activity.     

Possibility of Bacteroides gingivalis hemagglutinin possessing protease activity revealed by inhibition studies

Inhibition of hemagglutinin (HA) activity in a membrane fraction of Bacteroides gingivalis was examined using various compounds. Leupeptin and anti-pain inhibited the HA activity at nM order. This potency was lost when the aldehyde group of leupeptin was converted to an alcohol moiety. Irreversible protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK), p-chloromercuribenzoate (PCMB), and N-ethylmaleimide (NEM) were also inhibitory. From the inhibition experiments, we speculate that the HA possesses protease activity and that the same site of the molecule participates in the erythrocyte binding and the substrate binding.     

In vivo anti-tumor immunity detected by leukocyte adherence inhibition

Summary The immune response of mice to a transplacentally induced alveolar cell tumor was studied with the leukocyte adherence inhibition (LAI) assay. The lung tumor, designated 85, was induced in a C3HfB/HeN (C3Hf) mouse by l-ethyl-l-nitrosourea (ENU). While a dose of 10⁵ cells of this tumor does not grow in syngeneic C3Hf mice, it does grow readily in (A×C3Hf)F₁ hybrid mice. The tumor possesses a tumor associated transplantation antigen (TATA) which cross-reacts with a normal tissue alloantigen in strain A/HeN (A) mice. Normal mice, tumor-immunized C3Hf mice, and tumor-bearing (A×C3Hf)F₁ mice possessed peritoneal cells, the majority of which adhered rapidly to glass and resisted gentle washing. When incubated with an extract of the 85 tumor, peritoneal cells from tumor-immunized mice demonstrated marked inhibition of adherence (62.4%) compared to similarly incubated peritoneal cells of either normal mice (30.3%) or tumor bearing mice (37.1%). Specificity of the reactivity in the LAI assay was demonstrated with a neuroblastoma extract and peritoneal cells from neuroblastoma-immunized C3Hf mice. Peritoneal cells from lung tumor-immunized mice, but not tumor-bearing mice, responded to a lung extract from strain A mice. In contrast to the microcytotoxicity assay, the LAI assay is capable of distinguishing the effective anti-tumor response of tumor-immunized C3Hf mice from the ineffective immune response of tumor-bearing (A×C3Hf)F₁ mice.     

Structural aspects of the inhibition of DNase and rRNase colicins by their immunity proteins

Nuclease E colicins that exert their cytotoxic activity through either non-specific DNase or specific rRNase action are inhibited by immunity proteins in a high affinity interaction that gives complete protection to the producing host cell from the deleterious effects of the toxin. Previous X-ray crystallographic analysis of these systems has revealed that in both cases, the immunity protein inhibitor forms its highly stable complex with the enzyme by binding as an exosite inhibitor-adjacent to, but not obscuring, the enzyme active site. The structures of the free E9 DNase domain and its complex with an ssDNA substrate now show that inhibition is achieved without deformation of the enzyme and by occlusion of a limited number of residues of the enzyme critical in recognition and binding of the substrate that are 3' to the cleaved scissile phosphodiester. No sequence or structural similarity is evident between the two classes of cytotoxic domain, and the heterodimer interfaces are also dissimilar. Thus, whilst these structures suggest the basis for specificity in each case, they give few indications as to the basis for the remarkably strong binding that is observed. Structural analyses of complexes bearing single site mutations in the immunity protein at the heterodimer interface reveal further differences. For the DNases, a largely plastic interface is suggested, where optimal binding may be achieved in part by rigid body adjustment in the relative positions of inhibitor and enzyme. For the rRNases, a large solvent-filled cavity is found at the immunity-enzyme interface, suggesting that other considerations, such as that arising from the entropy contribution from bound water molecules, may have greater significance in the determination of rRNase complex affinity than for the DNases.     

Split immunity: immune inhibition of rat gliomas by subcutaneous exposure to unmodified live tumor cells

Gliomas that grow uninhibited in the brain almost never metastasize outside the CNS. The rare occurrences of extracranial metastasis are usually associated with a suppressed immune system. This observation raises the possibility that some gliomas might not grow outside the CNS due to an inherent immune response, We report in this study that the highly malignant F98 Fischer rat undifferentiated glioma, which grows aggressively in the brain, spontaneously regresses when injected live s.c. We found that this regression is immune-mediated and that it markedly enhances the survival or cures rats challenged with the same tumor intracranially either before or after the s.c. live-cell treatment. Adoptive transfer experiments showed the effect was immune-mediated and that the CD8 T cell fraction, which exhibited direct tumor cytotoxicity, was more effective than the CD4 T cell fraction in mediating resistance to intracranial challenge of naive rats. Brain tumors from treated rats exhibited enhanced CD3(+)CD8(+)CD4(-) and CD3(+)CD4(+)CD8(-) T cell infiltration and IFN-γ secretion. The results in the F98 glioma were corroborated in the Lewis rat CNS-1 astrocytoma. In both tumor models, s.c. treatment with live cells was significantly better than immunization with irradiated cells. We propose in this study a location-based immunotherapeutic phenomenon we term "split immunity": a tumor that thrives in an immune-privileged site may be inhibited by injecting live, unmodified tumor cells into a site that is not privileged, generating protective immunity that spreads back to the privileged site. Split immunity could explain several long-standing paradoxes regarding the lack of overt extracranial metastasis in patients with primary brain tumors.