Augmentation of natural killer cell activity in mice by Bru-Pel.

The immunomodulatory and anti-tumor activity of Bru-Pel, an aqueous-ether extracted residue of Brucella abortus (strain 456), was investigated. Bru-Pel was administered to C57BL/6 mice intraperitoneally (i.p.) and tested for its effect on natural killer (NK) cell activity in spleen cells, liver, and peritoneal cavity. Three days after injecting 100 micrograms of Bru-Pel i.p., the cytotoxicity of spleen cells against YAC-1 target cells, assessed by LU20 increased by approximately two-fold and nonparenchymal cells of liver by greater than six-fold. The highest stimulatory effect of Bru-Pel was seen with peritoneal exudate cells, and 47-fold augmentation of NK cell activity was observed. Bru-Pel treatment made spleen, liver, and peritoneal exudate cells capable of lysing P815 mastocytoma cells, a tumor cell line highly resistant to lysis by unstimulated NK cells. In vivo, Bru-Pel inhibited the formation of experimental BL6 melanoma metastases; however, there was no significant effect on the eradication of established pulmonary metastatic lesions. These results demonstrate that in addition to its previously described macrophage-activating ability, Bru-Pel is highly efficient in stimulation of NK cell-mediated cytotoxicity in mice.

Phylogenetic hierarchy of antigen-presenting ability in antigen-specific T cell activation.

The phylogenetic hierarchy of antigen-presenting ability was shown to exist in xenogeneic mouse, rat and human T cell-antigen-presenting cell (APC) interaction. The antigen-presenting ability of human APC is dominant over that of rat APC and the ability of rat APC is dominant over that of mouse APC. The HLA-DR molecule was shown to function for antigen presentation to PPD-specific autologous human and xenogeneic murine T cells.

Cellular FITC-linked immunospecific assay (cell-FLISA) for detection of monoclonal antibodies against cell-surface antigens.

A cellular fluorescein isothiocyanate (FITC)-linked immunospecific assay (Cell-FLISA) has been established using the recently developed fluorophotometer for microplates. In the Cell-FLISA system, monoclonal antibodies specific for the surface antigens of live cells are detected by measuring the fluorescence intensity of an FITC-labeled second antibody: goat anti-mouse immunoglobulin antibody. It takes only 2 min to count 96 samples in microplate wells using the fluorophotometer for microplates. Moreover, by this system, the analysis is finished within 2 hr. Thus, the Cell-FLISA system has advantages in screening a large number of samples, such as hybridoma cell lines secreting monoclonal antibodies against cell-surface antigens.

Interspecies cross-reactivity of Class II antigen of MHC determined by syngeneic, allogeneic and xenogeneic B and T cells.

Although this chapter ought to summarize the role of MHC antigens in T cell activation, the immunobiological meaning of the polymorphism of Class II antigens, as well as that of Class I antigens, is still unresolved. The antigen-presenting ability of human APC is dominant over that of murine APC in the stimulation of antigen-specific xenogeneic T cells. In addition, xenoreactive murine T cells specific for human PBL failed to recognize the polymorphic determinant of Class II antigens of human MHC. On the basis of the data, Class II antigens may be seen to have some role as antigen-presenting molecules rather than as restricting molecules, at least, in the xenogeneic APC-T cell interaction or the xenogeneic MLR responses. These data together with the fact that the linkage disequilibrium found among the various groups of alleles encoding Class I and II antigens making up an MHC haplotypes suggest that the MHC may play a key role during evolution. These studies using xenogeneic cell interaction may shed some light on the immunobiological function of polymorphism of MHC antigens in the mechanisms of T cell activation, and the evolutional history of the polymorphism of the NHC in self or not-self recognition by T cells.

Establishment of Epstein-Barr virus-associated lymphoma cell line SUBL with t(2;3)(p11;q27) from a liver transplant patient.

A new lymphoma cell line, designated SUBL, was established from a Japanese patient with Epstein-Barr virus (EBV)-associated lymphoma, which developed during FK 506 therapy after liver transplantation. This cell line has undergone 80 passages over a period of 22 months. The cultured cells were positive for CD19, CD20, CD21, CD22, CD23, and HLA-DR, and negative for CD10 and surface immunoglobulins. Immunoglobulin gene analysis revealed rearrangements of JH and JK. T-cell antigens or T-cell receptor gene rearrangements were not observed on the cell line. The SUBL cells were positive for Epstein-Barr virus nuclear antigen (EBNA). The EBV genome was detected in the original tissue and the cell line by the in situ hybridization method. These data indicate that this cell line represents the B-cell lineage at a pre-B-cell stage. SUBL cells showed successful heterotransplantation to mice with severe combined immunodeficiency (SCID). Chromosomal analysis revealed the karyotype 46,XY,t(2;3)(p11;q27). Molecular studies showed that c-myc, N-myc, and bcl-2 were not rearranged. This cell line will provide a useful in vitro system to study the relationship between chromosomal abnormalities and the activation of cellular oncogenes.

Formation of vesiculated large bodies of Staphylococcus aureus L-form in a liquid medium.

A method is described in which cells of Staphylococcus aureus can be converted to vesiculated large bodies of L-form. When coccal cells were incubated in a liquid growth medium containing D-cycloserine, N-acetylmuramidase and subtilisin, a large number of vesiculated large bodies were formed. Electron microscopy revealed that development of internal vesicles arose after 6 hr of incubation.. When growth inhibitory concentrations of rifampicin, novobiocin, or chloramphenicol were added to the culture at 6 hr of incubation, small-sized nonvesiculated bodies were produced instead of vesiculated forms. The viability of cultures was reduced by rifampicin and novobiocin but not by chloramphenicol.

Salt-induced L-forms of Staphylococcus aureus.

When growing cultures of a salt-sensitive strain of Staphylococcus aureus were inoculated on nutrient agar containing 0.8 M NaCl and 0.5% bovine serum albumin, typical colonies of L-form developed extensively after 2 days of incubation at 30 C. Incubation of growing cultures with lipoteichoic acid, sodium polyanethole sulfonate and subtilisin resulted in inhibition of L-form induction.

Amino acid transport in Mycobacterium smegmatis.

The transport of d-alanine, d-glutamic acid, and d-valine in Mycobacterium smegmatis was compared quantitatively with that of their l-isomers. It appeared that the uptake of d-alanine was mediated by an active process displaying saturation kinetics characteristic of enzyme function, whereas the uptake of d-glutamic acid was accomplished by a passive process showing diffusion kinetics. Both processes were involved in the uptake of l-alanine, l-glutamic acid, d-valine, and l-valine. d-Valine competed with l-valine for entry into the cell through a single active process. d-Alanine and l-alanine also utilized the same active process, but the d-isomer could not enter the cell through the passive process. The passive process exhibited characteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents and showed competition among structurally related amino acids. These last findings suggested that the passive process is a facilitated diffusion.

Protoplast formation of selected Mycobacterium smegmatis mutants by lysozyme in combination with methionine.

Lysozyme-sensitive mutants of Mycobacterium smegmatis, isolated by nitrosoguanidine treatment, have been converted into protoplasts in a nutritionally enriched medium containing lysozyme and DL-methionine.

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells.

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.

Salt-induced cell lysis of Staphylococcus aureus.

Cell lysis was efficiently induced in Staphylococcus aureus by the addition of 0.3 M NaCl to exponentially growing cultures at 30 degrees C. When cells harvested at the exponential phase were incubated in buffer with NaCl, autolysis occurred. Treatment with chloramphenicol failed to induce cell lysis by NaCl. The effects of NaCl on growing cells and harvested cells were inhibited by the addition of sodium polyanethole sulfonate, subtilisin, cardiolipin, and lipoteichoic acid. These agents diminished the activity of a cell wall-lytic enzyme liberated from the cells in the presence of NaCl. Lysis induced by salt appears to be catalyzed by a similar lytic enzyme in growing and harvested cells.

Protease-induced multicell formation in Staphylococcus haemolyticus.

Multicellular cells were efficiently induced in Staphylococcus haemolyticus by the addition of protease to exponentially growing cultures at 30 C. Electron microscopy revealed the formation of tetrad-shaped multicells that were septated but not separated from each other. Incubation of the multicells with extract from the cells grown without protease resulted in a fourfold increase in the number of colony-forming units as compared with the untreated control. An electrophoretic analysis showed that protease caused a loss of cell wall-lytic activity of the cell, which possibly led to the formation of multicells through cessation of cross wall separation.

Relationship between beta-lactamase activity and resistance to beta-lactam antibiotics in Mycobacterium smegmatis.

Penicillin-susceptible mutants and beta-lactamase-negative mutants were isolated from Mycobacterium smegmatis after nitrosoguanidine mutagenesis. Both the mutants were found to be susceptible to low levels of penicillin and cephalosporins by twofold dilution testing. Clavulanic acid reduced the minimal inhibitory concentrations of beta-lactamase-labile beta-lactams for the penicillin-susceptible mutants and the parent strain, but had no effect on the susceptibility of the beta-lactamase-negative mutants. Comparison of the beta-lactamase activities found in these mutants and the parent strain indicated that there was a rough correlation between the beta-lactamase level in these organisms and their susceptibility to beta-lactams.

Inhibition of growth of Mycobacterium smegmatis and of cell wall synthesis by D-serine.

d-Serine inhibited the growth of Mycobacterium smegmatis and induced the morphological alteration of the bacilli. The growth inhibitory action of d-serine was partially reduced by an equimolecular concentration of d-alanine. The combination of glycine with d-alanine reversed the growth inhibition produced by d-serine more than did d-alanine alone. In cells cultured in the presence of d-serine, the amounts of alanine, diaminopimelic acid, and glycine inserted into the cell wall mucopeptide were reduced, and serine was increased. The intracellular accumulation of a precursor of cell wall mucopeptide was increased by d-serine, and this accumulation was reduced by d-alanine. d-Serine competed with glycine for incorporation into the cell wall mucopeptide. The incorporation of l-aspartic acid into diaminopimelic acid residues in the cell wall mucopeptide was markedly inhibited by d-serine. Three mutants resistant to d-serine were isolated by nitrosoguanidine treatment. In these mutants the effects of d-serine on the sites of cell wall mucopeptide synthesis were all reduced. Thus, d-serine inhibition of the growth is due to replacement of glycine residues of the cell wall mucopeptide with d-serine and inhibition of the cell wall synthesis by blocking the formation of d-alanine and diaminopimelic acid.

Detection of active form of transforming growth factor-beta in cerebrospinal fluid of patients with glioma.

We examined transforming growth factor-beta (TGF-beta) activity in cerebrospinal fluid of 39 patients with various brain tumors, and found it in 10 glioma cases that had lesions related to subarachnoid space or ventricle. In one glioma case, TGF-beta detected on admission disappeared after radiation and chemotherapy. We confirmed that five glioma cell lines produced TGF-beta, and that four of them produced active form of TGF-beta directly. The active form of TGF-beta was also identified from cerebrospinal fluid before the acidification treatment in two cases. The calculated contents were 110 ng/ml and 18 ng/ml. These results indicate that active form of TGF-beta is directly produced by tumor cells in patients with glioma, and may contribute to immunodeficiency of the host.

Cyclosporine promotes the induction of thymic lymphomas in C57BL/6 mice initiated by a single dose of gamma-radiation.

We previously demonstrated that a single dose of gamma-radiation (350 rads) was able to induce thymic lymphomas in C57BL mice when followed by promoting treatment with oral cyclosporine (CsA), a non-genotoxic immunosuppressant. We have now tested the efficacy of various doses of gamma-radiation as an initiator of CsA promotion of the induction of thymic lymphomas in male C57BL mice. The effects of oral CsA on the splenic natural killer (NK) cell activity of non-irradiated and irradiated (400 rads, 1x) mice were tested by the standard 51Cr release assays against YAC-1 cells. The cumulative incidence of thymic lymphomas induced by a single dose of gamma-radiation at 100, 200, 400 and 600 rads were 10, 25, 63 and 75% respectively, after 42 weeks of CsA promotion. The splenic NK cell activity in non-irradiated mice given CsA for 4 weeks was twice as high as that in the control mice. CsA inhibited poly I:C-induced augmentation of the splenic NK cell activity. In mice given a single dose (400 rads) of gamma-radiation and CsA for 4 weeks, a similar but reduced enhancement of the splenic NK cell activity as seen in non-irradiated mice was observed. These results indicate that the efficacy of CsA promotion in the induction of thymic lymphomas is dependent on the initiating doses of gamma-radiation, and that CsA enhances host splenic NK cell activity during the early stage of tumor promotion.