Comparison of the murine humoral immune response to recombinant simian virus 40 large tumor antigen: Epitope specificity and idiotype expression

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag) was used to immunize inbred strains of mice to compare the humoral immune responses. Specifically we examined the epitope specificities and idiotype (Id) expression on anti-(SV40 T-Ag) responses induced in BALB/c and C57BL/6 inbred strains of mice. The predominant SV40 T-Ag epitopes recognized by the anti-(SV40 T-Ag) responses appeared to differ between these two inbred strains, this being based on the ability of sera to inhibit the binding of several murine monoclonal antibodies specific for SV40 T-Ag. In addition, anti-(SV40 T-Ag) responses produced in C57BL/6 mice failed to express a previously described cross-reactive Id expressed in the anti-(SV40 T-Ag) response in BALB/c mice. This cross-reactive Id is detected by a mouse monoclonal anti-Id, designated 58D, which has been shown to represent a potential focal point for manipulating the humoral immune response to SV40-induced tumors in BALB/c mice. Together, these data indicate that the functional duality of the humoral immune response, as assessed by epitope recognition and Id expression, differs between these two inbred strains of mice when immunized with a recombinant SV40 T-Ag.     

Examination of lymphokines induced in mice following immunization with recombinant simian virus 40 large tumor antigen

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was used to immunize BALB/c mice to examine the lymphokines produced following immunization. Specifically, we examined production of interleukin-2 (IL-2), IL-4, IL-5 and interferon (IFN) from immune lymphocytes cultured with decreasing concentrations of recombinant SV40 T-Ag. We identified elevated levels of IFN and IL-2 by enzyme-linked immunosorbent assay and a murine CTLL-2 proliferation biossay respectively. We were unable to detect either IL-4 or IL-5. These data indicate the previously reported tumor immunity induced by recombinant SV40 T-Ag immunization most likely reflects a TH1-like immune response based on thein vitro production of both IFN and IL-2 by immune lymphocytes.     

Nitrogen fixation efficiency,natural15N abundance,and morphology of mesquite (Prosopis glandulosa) root nodules

Summary The relative nitrogen fixation efficiencies (RE 1-[H₂ evolved÷C₂H₂ reduced]·100) of four mesquite (Prosopis glandulosa var.torreyana) rhizobia (Strains WR 1001, WR 1002, L5, L9) and a cowpea rhizobia (Strain 176A32) on mesquite were evaluated in a glasshouse experiment. Plant yield, shoot N accumulation, and the natural¹⁵N abundance (¹⁵N) of nodule tissue were determined. Strain WR 1002 failed to nodulate mesquite and strain L5 produced ineffective nodules. Among the three effective strains (WR 1001, L9, 176A32) the cowpea strain (176A32) and strain L9 had significantly higher RE than strain WR 1001. Differences in RE, however, were not accompanied by significantly higher plant yield and shoot N accumulation. The difference in¹⁵N abundance between foliar tissue and nodules (nodules minus leaves) was 0.47 ¹⁵N for the ineffective L5 nodules, while for the effective WR 1001, L9, and 176A32 nodules, respectively, this difference was 8.35, 7.81, and 8.35 ¹⁵N. This indicates a similar relationship between N₂-fixing effectiveness and natural¹⁵N enrichment of nodules that was previously observed in soybeans (Glycine max, L. Merr.). Strains WR 1001 and L9 produced elongate, indeterminate nodules typical for mesquite. The ineffective L5 nodules had few infected cells and an abundance of cortical amyloplasts. Mesquite nodules produced by the cowpea strain were spherical and were somewhat more similar in internal morphology to determinate nodules typical of cowpea than indeterminate nodules normally associated with mesquite.     

Fractionation of lymphocytes involved in the generation of cell-mediated cytotoxicity over insolubilized conjugated histamine columns.

Spleen cells from normal BALB/c mice were cultured in vitro with irradiated C57BL/6 stimulating cells. Five days later the T cell-mediated cytotoxic activity of the effector cells was assessed with a 51Cr-release assay that used H-2bEL-4 tumor cells as targets. Before the BALB/c responding lymphocytes were sensitized they were fractionated by passing the spleen cells over insolubilized histamine rabbit serum albumin Sepharose columns (H-RSA-S) or over rabbit serum albumin Sepharose (RSA-S) control columns. Fractionation of cells over the H-RSA-S columns depleted or significantly reduced the cytotoxic potential of the unretained cells. All cytotoxic potential was recovered when the cells that adhered to the H-RSA-S were eluted from the columns. In contrast, no effect on responsiveness was detected after the cells had been fractionated over the control column. The loss of response potential by the cells that did not adhere to H-RSA-S could not be accounted for by removal of macrophages nor by the concentration of cells with suppressor activity in the effluent. These cell fractionation studies raise the possiblity but do not prove that cytotoxic precursor cells may express amine receptors that could be responsible for their retention by insolubilized histamine columns.     

Preferential response patterns of cytotoxic T lymphocytes specific for fluorescein isothiocyanate-[FITC] modified autologous cells

Murine cytotoxic responses to TNP-modified syngeneic cells (TNP-self) have been shown to exhibit preferential recognition of K or D end self products encoded by the H-2 complex. In the present study, a number of B10 congenic and recombinant mouse strains were investigated to determine the H-2K and H-2D-restricted FTC-self CTL response patterns, and these were compared with the CTL response patterns obtained for TNP-self. The results indicate that for strains possessing the H-2k,d,h2,h4 haplotypes, respectively, preferential CTL responses were observed against FTC recognized in association with Kk over Dk, Dd over Kd, and Kk over Db. These patterns of preferential CTL responses were the same as those reported for TNP-self as well as several anti-viral CTL responses. In contrast to the results obtained in the B10.A strain, in which Kk preference was observed over Dd for TNP-self CTL, no preferential CTL response was observed when FTC was recognized in association with Kk and with Dd. In this context, it was observed that the CTL response to FTC recognized in association with Dd was particularly strong. This strong D end-associated response was shown to involve D locus products, and no evidence was obtained indicating that L locus self products were involved. These studies are discussed with respect to the possibility that different haptens can be recognized by CTL in association with different self determinants encoded by the same H-2 gene products.     

Involvement of calpain in diamide-induced cataract in cultured lenses.

Lenses cultured in diamide first developed outer cortical opacities followed by nuclear cataract. Lens hydration and total calcium were markedly increased by diamide. Proteolysis of crystallins were observed in nuclear cataract lenses. Calpain in the soluble fraction of lenses cultured with diamide was decreased, while calpain in the insoluble fraction was increased. Co-culture with E64d, an inhibitor of cysteine protease such as calpain, especially prevented nuclear opacities and proteolysis of crystallins, indicating that calpain was involved in cataract formation by diamide.     

Proline fed to intact soybean plants influences acetylene reducing activity and content and metabolism of proline in bacteroids

Supplying l-proline to the root system of intact soybean (Glycine max [L.] Merr.) plants stimulated acetylene reducing activity to the same extent as did supplying succinate. Feeding l-proline also caused an increase in bacteroid proline dehydrogenase activity that was highly correlated with the increase in acetylene-reducing activity. Twenty-four hours after irrigating with l-proline, endogenous proline content had increased in host cell cytoplasm and bacteroids, about three- and eightfold, respectively. In bacteroids, proline concentration was calculated to be at least 3.5 millimolar. In experiments in which [U-¹⁴C]l-proline was supplied to uprooted, intact plants incubated in aerated solution, ¹⁴C-labeled products of proline metabolism, as well as [¹⁴C]proline itself, accumulated in both host cells and bacteroids. When plants were incubated in aerated solutions containing [5-³H]l-proline, ³H-labeled proline was found in host cells and bacteroids. [³H] Pyrroline-5-carboxylate was found in bacteroids, but not host cells, after a 2-hour incubation in [5-³H]l-proline. When [U-¹⁴C]l-proline was supplied for 24 hours, a significant amount of [¹⁴C] pyrroline-5-carboxylate was found in the host cells, in contrast with the results from the shorter incubation in [5-³H]proline, although the amount in the host cells was only about half the quantity found in the bacteroids. Taken as a whole, these results indicate that proline crosses both plant and bacterial membranes under the in vivo experimental conditions utilized and are consistent with a significant role for proline as an energy source in support of bacteroid functioning. In spite of the increase in acetylene-reducing activity when proline was supplied to the root system of intact plants, proline application did not rescue stemgirdled plants from loss of acetylene-reducing activity, although succinate application did. This suggests a nonphloem route for succinate, but not proline, from roots to nodules.     

H2Kk can influence whether cytotoxic T lymphocytes recognize trinitrophenyl in association with H2Dk unique or H-2Kk and H-2Dk shared self determinants

The present study investigates the effect of trinitrophenyl- (TNP) modified H-2Kk (TNP-Kk) antigens on the generation of anti-TNP-Dk restricted cytotoxic T lymphocyte (CTL) responses. C3H.OH mice were primed to TNP-self by skin-painting with trinitrochlorobenzene, and spleen cells from these primed mice were subsequently stimulated in vitro with TNP-self. The effector cells generated exhibited appreciable lysis of TNP-modified C3H.OH blast target cells. Cold target inhibition studies demonstrated the generation of two effector cell populations: one that recognizes TNP in association with unique Dk self determinants, and one that recognizes TNP in association with self determinants shared between TNP-Kk and TNP-Dk. This was in contrast to primed C3H/He spleen cells, which did not generate CTL that recognized TNP in association with unique Dk self determinants. When spleen cells from (C3H/He x C3H.OH)F1 mice primed to TNP were stimulated in vitro with TNP-C3H.OH cells, unique Dk self determinants were recognized in association with TNP. However, in vitro stimulation of the same F1 responding cells with TNP-C3H/He or TNP-F1 cells failed to elicit CTL that utilized these Dk-unique self determinants. The findings of this study demonstrate that unique or shared H-2Dk determinants can be differentially utilized by CTL populations, depending on the H-2 alleles expressed by the stimulator cells.