Elevated intracellular dCTP levels reduce the induction of GC -> AT transitions in yeast by ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine but increase alkylationinduced GC -> CG transversions

The second sentence of the second paragraph on p. 463 (beginningat line 10) should read: ‘It seems unlikely that the enlargeddCTP pool in MGK-dp depressed cellular responses to the alkylatingagents simply by acting as a target for EMS and MNNG (Topaland Baker, 1982), so that effectively less mutagen attackedthe DNA.’     

Defects in base excision repair combined with elevated intracellular dCTP levels dramatically reduce mutation induction in yeast by ethyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine

Previously, we determined that elimination of deoxycytidylate (dCMP) deaminase (DCD1) in the yeast Saccharomyces cerevisiae increases the intracellular dCTP:dTTP ratio and reduces the induction of G·C → A·T transitions in the SUP4-o gene by ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Simultaneously, the G·C → C·G transversion frequency rises substantially. We attributed the first response to dCTP outcompeting dTTP for incorporation opposite O⁶-alkylguanine, and the second outcome to the increased dCTP pool causing error-prone repair of apurinic (AP) sites resulting from the removal or lability of N⁷-alkylguanine. To test the latter hypothesis, we used isogenic dcd1 strains deleted for either of two genes (MAG1: 3-methyladenine glycosylase; APN1: apurinic endonuclease) involved in the repair of N⁷-alkylguanine. In these backgrounds, EMS or MNNG induction of total SUP4-o mutations, G·C → A·T transitions and G·C → C·G transversions were reduced by >98%, >97%, and >80%, respectively. Mutation frequencies in the dcd1 apn1 strain were close to those for spontaneous mutagenesis in the wild-type parent. These findings argue that misincorporation of dCTP during repair of alkylation-induced AP sites is responsible for the increased G·C → C·G transversion frequency in the dcd1 strain treated with EMS or MNNG. The data also demonstrate that defective repair of AP sites coupled with an elevated dCTP:dTTP ratio eliminates most EMS and MNNG mutagenesis. In addition, the results point to a role for AP sites in the production of some EMS- and MNNG-induced G·C → A·T transitions as well as other substitutions in the dcd1 strain. Environ. Mol. Mutagen. 32:173–178, 1998 © 1998 Wiley-Liss, Inc.     

Glutathione S-transferase-mediated induction of GC → AT transitions by halomethanes in salmonella

Halomethanes are among the most common mutagenic and carcinogenic disinfection by-products present in the volatile/semivolatile fraction of chlorinated drinking water. Recent studies have demonstrated that the mutagenicity of dichloromethane (CH₂Cl₂) and bromodichloromethane (BrCHCl₂) can be mediated by a theta-class glutathione S-transferase (GSTT1-1). These studies used strain RSJ100 of Salmonella, which is a derivative of the base-substitution strain TA1535 (hisG46, rfa, δuvrB), into which has been cloned the GSTT1-1gene from rat. In the present report, we have ex tended these studies by demonstrating that the mutagenicity of two additional brominated trihalomethanes, bromoform (CHBr₃) and chlorodibromomethane (ClCHBr₂), are also mediated by GSTT1-1 in RSJ100. Using a Tedlar bag vaporization technique, the mutagenic potencies (revertants/ppm) for these two compounds as well as the compounds tested previously rank as follows: CHBr₃ ≈ ClCHBr₂ > BrCHCl₂ ≈ CH₂Cl₂. To explore the mutational mechanism, we determined the mutation spectra of all four halomethanes at the hisG46 allele by per forming colony probe hybridizations of ∼100 revertants induced by each compound. The majority (96–100%) of the mutations were GC → AT transitions, and 87–100% of these were at the second position of the CCC/GGG target. In contrast, only15% of mutants induced by CH₂Cl₂ were GC → AT transitions in the absence of the GSTT1-1 gene in strain TA100 (a homologue of TA1535 containing the plasmid pKM101). The ability of GSTT1-1 to mediate the mutagenicity of these di- and trihalomethanes and the induction of almost exclusively GC → AT transitions by these compounds suggest that these halomethanes are activated by similar pathways in RSJ100, possibly through similar reactive intermediates. The implications of these findings are discussed in relation to previous experimental work on the GST-mediated bioactivation of dihalomethanes, which includes the possible formation of GSH intermediates and/or GSH-DNAadducts. Environ. Mol. Mutagen. 30:440–447, 1997 Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and as such, is in the public domain in the United States of America.

     

Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure

Summary Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 ( haploid), P-540 (a/ diploid), and P-544 (a/ diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1x10^-1) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a// genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an / genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploids.     

Decrease of blood dendritic cells and increase of tissue‐infiltrating dendritic cells are involved in the induction of Sjögren's syndrome but not in the maintenance

We have demonstrated previously that, in primary Sjögren's syndrome (SS), immature myeloid dendritic cells (DCs) are decreased in blood and mature myeloid DCs are accumulated in salivary glands, suggesting recruitment of the myeloid DCs from blood to salivary glands. To verify whether this finding is universal in patients of not only primary SS but also secondary SS, in this study we analysed the blood DCs of secondary SS patients. We examined 24 secondary SS and 29 primary SS patients. A direct correlation between the decreased number of myeloid DCs and the duration of Sicca syndrome in primary and secondary SS was observed; namely, the reduction of myeloid DCs in blood was restored spontaneously with duration time of Sicca syndrome. We also examined the immunohistochemical staining of salivary glands of SS patients with monoclonal antibodies against fascin, CD11c and human leucocyte antigen DR (HLA‐DR). Fascin⁺ or CD11c⁺/HLA‐DR⁺ mononuclear cells were present in the salivary glands of secondary SS patients, as in primary SS. However, fascin⁺ mononuclear cells were barely detected in the salivary glands of a chronic phase of SS patients. We also found a negative correlation between the frequency of blood myeloid DCs and salivary gland‐infiltrating DCs in secondary SS patients, as well as primary SS. Our results suggest that the reduction of blood myeloid DCs and preferential trafficking of myeloid DCs into salivary glands is a common event in the early stage of SS. Myeloid DCs may play essential roles in the pathogenesis of Sicca syndrome of SS by initiating T helper cell immune responses.     

Vitamin A or E and a catechin synergize as vaccine adjuvant to enhance immune responses in mice by induction of early interleukin‐15 but not interleukin‐1β responses

Vitamins A and E and select flavonoids in the family of catechins are well‐defined small molecules that, if proven to possess immunomodulatory properties, hold promise as vaccine adjuvants and various therapies. In an effort to determine the in vivo immunomodulatory properties of these molecules, we found that although mucosal and systemic vaccinations with a recombinant HIV‐1_BaLgp120 with either a catechin, epigallo catechin gallate (EGCG) or pro‐vitamin A (retinyl palmitate) alone in a vegetable‐oil‐in‐water emulsion (OWE) suppressed antigen‐specific responses, the combination of EGCG and vitamin A or E in OWE (Nutritive Immune‐enhancing Delivery System, NIDS) synergistically enhanced adaptive B‐cell, and CD4⁺ and CD8⁺ T‐cell responses, following induction of relatively low local and systemic innate tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6) and IL‐17, but relatively high levels of early systemic IL‐15 responses. For induction of adaptive interferon‐γ and TNF‐α responses by CD4⁺ and CD8⁺ T cells, the adjuvant effect of NIDS was dependent on both IL‐15 and its receptor. In addition, the anti‐oxidant activity of NIDS correlated positively with higher expression of the superoxide dismutase 1, an enzyme involved in reactive oxygen species elimination but negatively with secretion of IL‐1β. This suggests that the mechanism of action of NIDS is dependent on anti‐oxidant activity and IL‐15, but independent of IL‐1β and inflammasome formation. These data show that this approach in nutritive vaccine adjuvant design holds promise for the development of potentially safer effective vaccines.     

IL‐18, but not IL‐15, contributes to the IL‐12‐dependent induction of NK‐cell effector functions by Leishmania infantum in vivo

Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK‐cell response in mice that required TLR9‐positive myeloid DC and IL‐12, but no IFN‐α/β. Here, we investigated whether IL‐15 or IL‐18 mediate the activity of IL‐12 or function as independent activators of NK cells. In contrast to earlier studies that described IL‐15 as crucial for NK‐cell priming in response to TLR ligands, the expression of IFN‐γ, FasL, perforin and granzyme B by NK cells in L. infantum‐infected mice was completely preserved in the absence of IL‐15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN‐γ secretion, cytotoxicity and FasL expression of NK cells from infected IL‐18^?/? mice were significantly reduced compared with controls, but, unlike IL‐12, IL‐18 was not essential for NK‐cell effector functions. Part of the NK‐cell‐stimulatory effect of IL‐12 was dependent on IL‐18. We conclude that IL‐15 is not functioning as a universal NK‐cell priming signal and that IL‐18 contributes to the NK‐cell response in visceral leishmaniasis. The cytokine requirements for NK‐cell activation appear to differ contingent upon the infectious pathogen.     

The absence of IFN-gamma leads to increased Th2 responses after influenza A virus infection characterized by an increase in CD4+ but not CD8+ T cells producing IL-4 or IL-5 in the lung

The cytokine IL-4 has been shown to be responsible for the switch of both CD4+ and CD8+ T cells to a Th2 or TC2 functional phenotype in vitro which both secrete IL-4 after stimulation. In contrast the presence of IFN-gamma interferes with the generation of Th2 and TC2 cells in vitro. Furthermore, it is well established that in the absence of IFN-gamma and the presence of IL-4 Th2 cells also develop in vivo. However, little is known about the conditions leading to the generation of TC2 cells in vivo. For this reason we investigated if Th2 and TC2 cells develop in the lung of IFN-gamma deficient mice which were infected with Influenza A virus. Surprisingly, we were only able to detect Th2 but not TC2 cells in the bronchoalveolar fluid and the mediastinal lymphnodes of IFN-gamma deficient mice infected with influenza A virus 1, 2 and 3 weeks by intracellular FACS staining for IL-4 or IL-5. In infected and uninfected wild type mice and uninfected IFN-gamma deficient mice we were not able to detect any Th2 or TC2 cells. These findings suggest that the prerequisites for Th2 and TC2 cell development are different in vivo than in vitro and may also explain why Th2 cells are more readily detected after immunisations or infections than TC2 cells in vivo.     

Blockade of CD28/B7 co-stimulation by mCTLA4-Hγ1 inhibits antigen-induced lung eosinophilia but not Th2 cell development or recruitment in the lung

We have studied the role of the CD28/B7 co-stimulatory pathway in the development of a Th2-type lung immune response. Mice injected two or three times intraperitoneally with ovalbumin in alum adjuvant and then re-exposed to the same antigen by intranasal (i.n.) inoculation show infiltration of the lung tissue and appearance in the broncho-alveolar lavage (BAL) fluid of significant numbers of eosinophils and lymphocytes, in a pattern which is reminiscent of asthmatic inflammation. The accumulation of eosinophils in the airways is completely dependent on interleukin (IL)-5 secretion by CD4⁺ T cells. We have used mice transgenic for a soluble form of murine CTLA-4 (mCTLA4-Hγ1) which binds to B7 molecules on antigen-presenting cells, thereby preventing their interaction with T cell-expressed CD28. mCTLA4-Hγ1-transgenic mice immunized intraperitoneally and challenged i.n. with ovalbumin failed to generate any eosinophil infiltration, suggesting that little or no IL-5 was secreted in the lungs of these mice. In contrast with the complete lack of eosinophils, the numbers and phenotypes of infiltrating lymphocytes were comparable in the lungs of mCTLA4-Hγ1-transgenic and normal mice. Also, lung lymphocytes from immunized mCTLA4-Hγ1-transgenic and normal mice could be shown to secrete comparable amounts of IL-4 and IL-5 when stimulated in culture in the absence of mCTLA4-Hγ1. We conclude that mCTLA4-Hγ1 can efficiently block the production of IL-5 during in vivo responses and inhibit eosinophil recruitment, but that it does not block the development of CD4⁺ T cells into Th2 cells with the potential to secrete IL-5.     

Modification of the anti-CD3-induced cytokine release syndrome by anti-interferon-γ or anti-interleukin-6 antibody treatment: Protective effects and biphasic changes in blood cytokine levels

Anti-interferon-γ (IFN-γ) antibodies were found to protect mice against pathological changes induced by injection of anti-CD3 antibody: incidence of diarrhea, severity of hypothermia and mortality rates were dramatically reduced. In anti-IFN-γ antibody-treated mice, IFN-γ blood levels were significantly reduced at 1.5 h post anti-CD3 challenge, but more elevated levels were found from 4 to 24 h. This rebound-like IFN-γ response coincided with more profound hypoglycemia. Tumor necrosis factor and interleukin (IL)-6 levels were not affected by anti-IFN-γ treatment. Exogenous IFN-γ, administered within 3 h (but not later) of the anti-CD3 challenge made the syndrome worse. Furthermore, inter-mouse strain differences in sensitivity to the anti-CD3 syndrome correlated with the ability of the strain to produce IFN-γ. Anti-IL-6 antibodies provided only marginal protection against hypothermia and mortality, but did markedly reduce hypoglycemia. Levels of biologically active IL-6 in serum were not influenced by anti-IL-6 antibody treatment during the first few hours after anti-CD3 challenge, but were significantly increased at later times. The data provide evidence that endogenous IFN-γ is a critical element in the early phase of the anti-CD3 syndrome; endogenous IL-6, while possibly being involved in hypoglycemia, seems of lesser importance for the outcome of the syndrome.     

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR β locus is incomplete but thymocyte development is not restored by TcR β or TcR αβ transgenes

The protein tyrosine kinase, p56^lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56^lck expression (p56^lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4⁺CD8⁺ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56^lck-/- mice and found an increase in the proportion of the CD44^?CD25⁺ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) β expression, may be delayed in the absence of p56^lck expression. In addition, the expression of a transgenic TcR β chain or TcR αβ pair did not restore thymic development in p56^lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56^lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56^lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56^lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one Vβ chain in TcR transgenic p56^lck-/- mice, we found that inhibition of endogenous TcR β gene rearrangement was almost complete in thymocytes of Vβ transgenic p56^lck-/- mice and we could not detect any peripheral T cells that expressed more than one Vβ chain in non-transgenic p56^lck-/- mice.