Effect of the calcium phosphate-mediated RNA uptake on the transfer of cellular immunity of a synthetic peptide of HIV-1 to human lymphocytes by exogenous RNA.

It is known that exogenous RNA molecules can be taken up by eukaryotic cells and can exert a variety of biological effects both in vitro and in vivo. The modulation of human lymphocytes by exogenous RNAs has medical implications. The exogenous RNA used in this study was obtained from lymphoid organs of animals immunized with the synthetic peptide p12 of HIV-1 and was referred to as p12-RNA. Human lymphocytes were transfected with the p12-RNA and the transfer of immunoreactivity of p12 was assessed by the lymphocyte proliferation and the leukocyte adherence inhibition assays. Our results indicate that the transfer of cellular immune response to the p12 occurred in 9 donors (60%) who were named responsive individuals whereas 6 donors (40%) were non-responsives. We also found that the calcium phosphate-mediated RNA uptake method is effective in converting non-responsive into responsive donors. The calcium phosphate-mediated RNA uptake may also be used to increase the efficiency of RNA transfection in other models with medical implications and to contribute to a better understanding of the molecular events involved in the uptake of RNA. Our findings give support for the use of exogenous RNAs obtained from lymphoid organs of immunized animals with synthetic peptides of HIV-1 in the immune reconstitution of individuals infected with HIV-1.

Immunotherapy of metastases with lymphocytes treated with exogenous RNA in mice bearing B16 melanoma.

Subline B16-F10, a variant cell line of B16 melanoma, is highly metastatic to the lung when injected intravenously into C57BL/6 mice. This experimental metastasis model was used to test the anti-tumor effect of exogenous RNA extracted from the lymphoid organs of immunized animals with B16-F10 cells. This RNA preparation is referred to as B16-RNA. Adoptive immunotherapy with lymphocytes treated with B16-RNA was effective in reducing significantly the number of pulmonary metastatic nodules. Lymphocytes incubated with medium alone or with RNA from non-immunized animals (N-RNA) were used as controls. The ability of B16-RNA in modulating antimetastatic activity of normal lymphocytes is abolished by hydrolysis with KOH. This finding indicates that the integrity of the polynucleotide chain is essential for the activity of B16-RNA. The anti-tumor effect of lymphocytes treated with B16-RNA was enhanced by incubation with a low dose of interleukin-2 (IL-2). A possible role of the double-stranded RNA dependent protein kinase in this phenomenon is discussed.

Transformation of Aspergillus nidulans by RNA from rat macrophages stimulated with lipopolysaccharide.

Exogenous RNA molecules can be incorporated into eukaryotic cells and can exert a variety of biological effects. We have previously described a model system for correcting genetic alterations of an Aspergillus nidulans mutant using homologous RNA and this phenomenon was named retrotransformation. In the present study, the retrotransformation of A. nidulans was performed with heterologous RNA which was extracted from rat macrophages stimulated with lipopolysaccharide (LPS). Protoplasts of A. nidulans were treated with this mammalian RNA and retrotransformants were detected by their ability to secrete the neutrophil recruitment inhibitory factor (NRIF) which is released by LPS-stimulated macrophages. Twenty two retrotransformant colonies were analyzed and only two retrotransformants, named RT1 and RT2, were able to secrete NRIF. The occurrence of sectors (RT1.1, RT2.1 and RT2.2) in retrotransformants RT1 and RT2 is due to mitotic instability which can be accompanied by loss of genomic extra-segments. The three sectors detected did not exhibit NRIF activity possibly due to loss of the NRIF gene present in the genome of retrotransformants RT1 and RT2. The NRIF like material secreted by RT2 shows the same lectin property and biological activity of NRIF released by LPS-stimulated macrophages. To date, this work is the first example of retrotransformation described in lower eukaryotes with heterologous RNA.

Anti-tumor effect of splenocytes treated with RNA from animals immunized with bovine myelin basic protein.

Chemically and immunologically, myelin basic protein (MBP) is very similar with the basic protein extracted from animal and human tumors. The results of this study demonstrated that splenocytes from C57BL/6 mice bearing B16 melanoma cells are sensitized to MBP, suggesting that this protein may share common antigenic determinants with antigens from B16 melanoma cells. The RNA preparations isolated from lymphoid tissues of normal or immunized guinea pigs with bovine MBP are referred to as N-RNA or MBP-RNA, respectively. It was also found that MBP-RNA is active in transferring MBP reactivity to normal splenocytes whereas N-RNA had no effect. To investigate whether this transfer to MBP immunoreactivity could result in a protective immunity, C57BL/6 mice transplanted with B16 melanoma received normal splenocytes treated with N-RNA or MBP-RNA. Two weeks after injection of B16-F10 cells, the mice were sacrificed and the tumor of each animal was removed and weighed. A significant inhibition of B16 melanoma growth was only achieved in C57BL/6 mice treated by splenocytes incubated with MBP-RNA which acts as an anti-tumor RNA. In this context, MBP could be considered as a tumor antigen.

Partial characterization of the RNA from LPS-stimulated macrophages that induces the release of chemotactic cytokines by resident macrophages.

It is well established that exogenous RNA is incorporated into eukaryotic cells and is able to exert various biological responses. Little, however, is known about the effects of such RNA on macrophages. In this study, we demonstrate that RNA extracted from macrophages stimulated with Escherichia coli lipopolysaccharide (LPS), referred to as L-RNA, in contrast to RNA from non-stimulated macrophages (N-RNA), induces the release of a macrophage-derived neutrophil chemotactic factor (MNCF) and interleukin-8 (IL-8) from macrophage monolayers. The effect of L-RNA was dependent of the integrity of the polynucleotide chain and was not due to LPS contamination since its ability to induce MNCF and IL-8 release was strongly reduced by RNase but was not affected by DNase or polymyxin B. The poly A(+)L-RNA and poly A(-)L-RNA fractions were able to induce the release of MNCF and IL-8, indicating that the L-RNA could be acting at transcriptional and translational levels. The demonstration that actinomycin-D and cycloheximide inhibited the release of MNCF and IL-8 by L-RNA-stimulated macrophages confirms this assumption. Fractionation of the total L-RNA by centrifugation on a 5-20% sucrose gradient showed that the L-RNA which sediments in the 4-5S region of the gradient is the only fraction capable of inducing the release of MNCF from naive macrophages. We have previously shown that macrophage monolayers stimulated with interleukin-1 beta or LPS release a low molecular RNA which also sediments in the same 4-5S region. Taken together, these results support our proposal that resident macrophages, when activated by injurious stimuli, in addition to secreting cytokines, also release a low molecular weight (4-5S) RNA which may act on the surrounding macrophages to further stimulate the release of cytokines. This process would amplify the inflammatory response and would increase the mechanisms involved in the defense response or tissue injury.

RNA-mediated transfer of cellular immunity to a synthetic env antigen of the human immunodeficiency virus (HIV-1).

A sheep was immunized with a synthetic peptide corresponding to amino acid residues 586-606 of the precursor envelope protein GP-160 of the HIV-1 including a conserved epitope region of the GP-41 transmembrane protein in the mature viral particles, referred to as SM 284 HIV-1 [1]. It is demonstrated that immune RNA extracted from the lymphoid organs of the immunized animal (SM 284 HIV-1 I-RNA) was able to transfer immune cellular reactivity to SM 284 HIV-1 in vitro to human and rabbit lymphocytes and in vivo to Cebus apella monkeys. The transfer was detected by the leukocyte adherence inhibition test (LAI) as an indicator of cellular reactivity. One of the most relevant results was the demonstration that SM 284 HIV-1 I-RNA was able to induce cellular immunological memory in vivo in monkeys. These results may be relevant to delineate a new alternative for immunomodulation against HIV infection.

Cellular immunity to bovine myelin basic protein in rats bearing malignant fibrous histiocytoma.

T lymphocytes from rats with malignant fibrous histiocytoma at different stages of tumor growth were investigated for sensitization to bovine myelin basic protein (MBP). The immunological reactivity was assessed by a leukocyte adherence inhibition assay as an in vitro correlate of cellular immunity. We found that only T lymphocytes obtained 4 and 6 days after tumor transplantation are reactive to MBP (1-2 micrograms/ml). Since the sequence of the antigenic determinants from MBP is known, our findings may make it possible to select synthetic epitopes for generating cytotoxic T lymphocytes to these tumor cells.

Liver trophism in dogs made diabetic by total pancreatectomy or alloxan administration.

1. In order to evaluate hepatic trophism in diabetic dogs, an experimental study was carried out on 30 adult mongrel dogs of both sexes weighing 11.9 +/- 1.6 kg, divided into three groups: 1) Controls (C) (N = 10), submitted to partial (30%) hepatectomy; 2) PD Animals (N = 10), submitted to partial hepatectomy plus total pancreatectomy with preservation of the duodenum during the same surgical intervention; 3) and AD Animals (N = 10), submitted to partial and simultaneously made diabetic with alloxan. 2. The animals were submitted to an oral glucose tolerance test (OGTT) one day before and on the 7th day after partial hepatectomy to evaluate the severity of diabetes. During the post-hepatectomy period the fasting glycemia values were similar for both diabetic groups (greater than 200 mg%). During OGTT, blood glucose levels of the diabetic groups peaked at 60 min but were significantly higher for the AD than for the PD group. The difference persisted at 120 and 180 min, but was no longer statistically significant. 3. Liver trophism was evaluated by measuring liver RNA content and the nuclear volume of hepatocytes. Both diabetic groups showed significantly lower RNA contents and absence of nuclear hypertrophy compared to partially hepatectomized controls probably because of the severe diabetes induced in these animals.

Transfer of cell-mediated immunity to HIV antigens in vivo to a Cebus monkey and in vitro to human lymphocytes with xenogeneic immune RNA.

1. Immune RNA (iRNA) was extracted from the spleen of a sheep immunized with human immunodeficiency virus (HIV) antigens. 2. The transfer of cell-mediated immunity to HIV antigens was accomplished by injecting iRNA into a Cebus monkey, as evaluated in vitro by leukocyte migration inhibition. The in vitro treatment of normal human leukocytes with iRNA also promoted the inhibition of leukocyte migration in the presence of HIV antigens. 3. These findings have important theoretical and potential practical applications in the field of Acquired Immunodeficiency Syndrome (AIDS).

Radioautographic and biochemical studies of secretion of venom protein in the South American rattlesnake Crotalus durissus terrificus.

Protein secretion was investigated in the main venom gland of the South American rattlesnake, using radioautographic and biochemical techniques after a single intracardiac injection of L-(3,5-3H)tyrosine. All the snakes were injected at the fourth day of the secretory cycle and killed at 1/2, 1, 2, 4, 8 and 24 hours after injection. Most of the radioactive amino acid is cleared from the blood stream up to four hours after injection. On the other hand the specific activity (c.p.m./mg of protein) of the intracellular proteins reaches a peak at the 4-hour time interval decreasing afterwards. There was a good correlation between the values of the specific activity of the intracellular proteins and those of the silver grain density over the secretory cells at the several time intervals after the injection of 3H-tyrosine. The results of the quantitative analysis carried out in light- and electron-microscope radioautographs led to the conclusion that venom proteins are synthesized in the rough endoplasmic reticulum of the secretory cells, transferred to the Golgi apparatus from where they are carried to the secretory tobule lumen by the secretion granules. The fact that the values of the relative concentration of the radioactivity of he intracisternal granules double at the last three time intervals, strongly suggests that these structures are formed by the aggregation of the amorphous material present inside the cisternae of the rough endoplasmic reticulum.