Properties of reticulum cell sarcomas in SJL/J mice

While T cells from SJL and from F₁ hybrids of SJL that do not express I-E antigens give strong proliferative responses to RCS, T cells from F₁ hybrids expressing surface I-E do not. The nature of the stimulating antigen on the RCS cell surface was examined using monoclonal antibodies. Complete inhibition of the T-cell proliferative response was obtained with antibodies to I-A antigens, whereas antibodies to I-E antigens did not inhibit at all. This inhibition was mediated via an effect of the antibodies on the stimulating cells. Biochemical characterization of immunoprecipitated ¹²⁵I- and 's S-labeled RCS antigens was performed using two-dimensional gel electrophoresis. Using this technique, I-A antigens were readily detected. However, neither Ia.7-specific antibodies nor antibodies specific for E_α : E _β complexes precipitated any E alpha or E beta chains. Comparison of I-A antigens from RCS and normal SJL spleen cells revealed minor mobility differences in the gels, possibly due to differences in glycosylation, the significance of which needs to be further evaluated. Examination of RNA extracted from RCS, using E alpha and A alpha cDNA probes showed that RCS cells do not transcribe the E alpha gene as has been shown previously for normal H-2 ^s cells. Furthermore, DNA from RCS cells showed a defect in the E alpha gene similar to that known to exist in normal H-2 ^s cells. Our findings exclude the presence of E alpha on RCS cells and suggest a major role for I-A, either alone or in conjunction with another as yet unidentified cell surface antigen, in the stimulation of T cells.     

Carcinogenesis in laboratory mice after low doses of ionizing radiation

Experimental data on the incidence of solid tumors from various long-term mouse studies performed at the Casaccia laboratories over several years were reconsidered, limiting the analysis to the results available for doses equal to or less than 17 cGy of neutrons and 32 cGy of X rays since these dose limits are reasonably close to the generally accepted low-dose levels for high- and low-LET radiation (i.e. D(high-LET) < 5 cGy and D(low-LET) < 20 cGy, respectively). The following long-term experiments with BC3F1 mice were reviewed: (a) females treated with single doses of 1.5 MeV neutrons or 250 kVp X rays, (b) males treated with fractionated doses of fission neutrons, and (c) mice of both sexes irradiated in utero 17.5 days post coitus with single doses of fission neutrons or X rays. An experiment with CBA mice of both sexes treated with single doses of fission neutrons was also included in this study. Analysis was done on animals at risk; thus all incidences of tumor-bearing animals were expressed as the percentage excess incidence with respect to the controls. Ovarian tumors and other solid neoplasms were considered. The percentage frequencies and mean survival times of tumor-free mice were also recalculated. The results indicate the existence of a region at low doses where the final incidence of solid neoplasms is indistinguishable from the background incidence. These data reinforce the idea that at low doses the effectiveness of ionizing radiation in inducing solid neoplasms in laboratory mice is very low.     

Blood kinetics of four intraperitoneally administered therapeutic candidate bacteriophages in healthy and neutropenic mice

Due to multiple-drug resistant bacteria, phage therapy is being revisited. Although most animal experiments focus on therapeutic efficacy, the blood clearance kinetics of phages have not been well described. For further development of an efficient therapeutic strategy, information on phage blood kinetics is important. In this study, time-course concentration changes in peripheral blood of healthy and neutropenic mice were measured using four therapeutic phages (φMR11, KPP10, φEF24C, and KEP10). The results showed a two- to three-day rapid phage clearance, which fits a two-compartment model.     

IL-33/ST2 contributes to severe symptoms in Plasmodium chabaudi-infected BALB/c mice

It has been reported that IL-33 contributes to potentiation of Th2 inflammatory diseases and protection against helminth infection. Increased plasma IL-33 levels have been observed in patients with severe falciparum malaria, however, the role of IL-33 in malaria remains unclear. Here we report that IL-33 enhances inflammatory responses in malaria infection. ST2-deficiency altered severity of inflammation in the liver and serum levels of pro-inflammatory cytokines such as TNF-α and IL-6, and IL-13 that is a Th2 cytokine during Plasmodium chabaudi infection. IL-13-deficient mice have similar phenotype with ST2-deficient mice during P. chabaudi infection. Furthermore, ST2- and IL-13-deficiency reduced mortality from P. chabaudi infection. These results indicate that IL-33/ST2 can induce production of proinflammatory cytokines, such as TNF-α and IL-6, through production of IL-13 in P. chabaudi-infected BALB/c mice, suggesting that IL-33/ST2 play a critical role in inflammatory responses to malaria infection. Thus, these findings may define a novel therapeutic target for patients with severe malaria.     

Double-stranded RNA induces similar pulmonary dysfunction to respiratory syncytial virus in BALB/c mice

Both respiratory syncytial virus (RSV) and influenza A virus induce nucleotide/P2Y purinergic receptor-mediated impairment of alveolar fluid clearance (AFC), which contributes to formation of lung edema. Although genetically dissimilar, both viruses generate double-stranded RNA replication intermediates, which act as Toll-like receptor (TLR)-3 ligands. We hypothesized that double-stranded RNA/TLR-3 signaling underlies nucleotide-mediated inhibition of amiloride-sensitive AFC in both infections. We found that addition of the synthetic double-stranded RNA analog poly-inosinic-cytidylic acid [poly(I:C)] (500 ng/ml) to the AFC instillate resulted in nucleotide/P2Y purinergic receptor-mediated inhibition of amiloride-sensitive AFC in BALB/c mice but had no effect on cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) transport. Poly(I:C) also induced acute keratinocyte cytokine-mediated AFC insensitivity to stimulation by the β-adrenergic agonist terbutaline. Inhibitory effects of poly(I:C) on AFC were absent in TLR-3(-/-) mice and were not replicated by addition to the AFC instillate of ligands for other TLRs except TLR-2. Intranasal poly(I:C) administration (250 μg/mouse) similarly induced nucleotide-dependent AFC inhibition 2-3 days later, together with increased lung water content and neutrophilic inflammation. Intranasal treatment of BALB/c mice with poly(I:C) did not induce airway hyperresponsiveness at day 2 but did result in insensitivity to airway bronchodilation by β-adrenergic agonists. These findings suggest that viral double-stranded RNA replication intermediates induce nucleotide-mediated impairment of amiloride-sensitive AFC in both infections, together with β-adrenergic agonist insensitivity. Both of these effects also occur in RSV infection. However, double-stranded RNA replication intermediates do not appear to be sufficient to induce either adenosine-mediated, CFTR-dependent Cl(-) secretion in the lung or severe, lethal hypoxemia, both of which are features of influenza infection.     

T cell potentiation in normal and autoimmune-prone mice after extended exposure to low doses of ionizing radiation and/or caloric restriction

In order to better understand the apparent physiologic up-regulation in response to low levels of potentially lethal insults, murine T lymphocytes were analysed for functional and phenotypic alterations after exposure to 0.005 Gy/day, 0.01 Gy/day and 0.04 Gy/day in groups of ad-libitum-fed and calorie-restricted mice. These studies were conducted in two strains of mice: the long-lived and immunologically normal C57Bl/6 +/+ and the congenic short-lived immunologically depressed C57Bl/6 lpr/lpr. Whole-body exposure to 0.01 Gy/day and 0.04 Gy/day for an extended period of 20 days was associated with an increase in splenic proliferative response and with shifts in the proportions of T cell subpopulations in the thymus and spleen of both strains. Caloric restriction independently altered functional activity and T cell subpopulations in the same direction as low dose rates of ionizing radiation. Although the dose-response augmentation in proliferative activity was similar in the two strains, observed alterations in thymic and splenic T cell subpopulations were clearly different, suggesting that different mechanisms were responsible for immune enhancement in each strain.     

NaCl-preferring NZB/B1NJ mice and NaCl-avoiding CBA/J mice have similar amiloride inhibition of chorda tympani responses to NaCl

Integrated chorda tympani nerve responses to NaCl were studied in two mouse strains, an NaCl-preferring NZB/B1NJ and an NaCl-avoiding CBA/J. The NaCl responses of both strains had similar magnitude and were suppressed by amiloride to a similar extent. This suggests that peripheral gustatory responsiveness to NaCl is not the only mechanism underlying mouse strain variation in NaCl acceptance.      

Response of hair types in mice to higher doses of x-rays

Doses of x-rays exceeding 1,000 r delivered to resting hair follicles of mice cause a variable greying response ranging from 60 to 100%. The purpose of this study was to study this phenomenon with specific reference to the response of zigzags and overhairs to irradiation. Accordingly, mice were exposed to 1,200 r and 1,300 r of x-rays and the response of hairs from a known area was compared with that of unirradiated controls and mice given 600 r, 800 r and 1,000 r, of radiation. It was found that at the higher doses, variations in total greying response were paralleled by variations in the response of both zigzags and overhairs, that there is relatively greater epilation of overhairs, and that zigzags contribute most heavily to both the white and the totally pigmented fractions. The results of this study therefore do not support the previous suggestion that the variable greying response is a function of a relative increase of pigmented overhairs in conjunction with selective damage of zigzags.     

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.     

Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays

We have shown previously that irradiations of mice with 0.1 or 0.2 Gy of X-rays stimulate anti-tumour cytotoxic activities of peritoneal macrophages and splenocytes enriched for NK lymphocytes and suppress the development of pulmonary tumour colonies. The up-regulated cytotoxicities were related to the production of nitric oxide by macrophages, and perforin and Fas ligand by the splenocytes, but specific blockade of these pathways did not totally suppress the effector cell-mediated cytolysis of the tumour target. Hence, other factors such as cytotoxic/cytostatic cytokines might have been produced by the effector cells. To test this possibility peritoneal macrophages and splenocytes were isolated from BALB/c mice which had been either once or tentimes whole body-irradiated with the total doses of 0.1 and 0.2 Gy of X-rays and assayed for the levels of IL-1beta, IL-2, IL-12, IFN-gamma and TNF-alpha in the incubation medium using the respective ELISA kits. The results demonstrate that both single and multiple exposures to the two low doses of X-rays significantly stimulate secretion of IL-1beta, TNF-alpha and IL-12 by macrophages and IL-2 and IFN-gamma by splenocytes, but the kinetics and magnitude of the induced changes in the production of these cytokines differ between the two irradiation protocols.     

Proliferative and anti-proliferative effects of retinoic acid at doses similar to endogenous levels in Leydig MLTC-1/R2C/TM-3 cells

Background

Vitamin A is suggested to be protective against oxidative stress. However, different authors observed pro-oxidant effects of retinoids both in experimental works and clinical trials. These discordances are the bases for the investigation of the proliferative and anti-proliferative properties of retinoic acid (RA) in biological systems.

Methods

Cell viability is determined with the MTT assay. Oxidative stress parameters are detected measuring catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. FABP5 mRNA levels are measured by RT-PCR. Autophagy and apoptosis are analyzed by Monodansylcadaverine (MDC) staining and TUNEL assay, respectively.

Results and conclusions

RA, at nutraceutic/endogenous doses (10–200 nM), increases cell viability of testes tumor Leydig cell lines (MLTC-1 and R2C) and modulates antioxidant enzyme activities, as CAT and GST. RA is able to induce proliferation through non-classical and redox-dependent mechanisms accompanied by increased levels of FABP5 mRNA. The redox environment of the cell is currently thought to be extremely important for controlling either apoptosis or autophagy. Apoptosis occurs at pharmacological doses, while autophagy, which plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis, is triggered at the critical concentration of 500 nM RA, both in normal and tumoral cells. Slight variations of RA concentrations are evaluated as a threshold value to distinguish between the proliferative or anti-proliferative effects.

General significance

Although retinoids have a promising role as antineoplastic agents, physiological levels of RA play a key role in Leydig cancer progression, fostering proliferation and growth of testicular tumoral mass.