Innate Lymphoid Cells Groups 1 and 3 in the Epithelial Compartment of Functional Human Intestinal Allografts.

We examined intraepithelial lymphocytes (IELs) in 213 ileal biopsies from 16 bowel grafts and compared them with 32 biopsies from native intestines. During the first year posttransplantation, grafts exhibited low levels of IELs (percentage of CD103(+) cells) principally due to reduced CD3(+) CD8(+) cells, while CD103(+) CD3(-) cell numbers became significantly higher. Changes in IEL subsets did not correlate with histology results, isolated intestine, or multivisceral transplants, but CD3(-) IELs were significantly higher in patients receiving corticosteroids. Compared with controls, more CD3(-) IELs of the grafts expressed CD56, NKp44, interleukin (IL)-23 receptor, retinoid-related orphan receptor gamma t (RORγt), and CCR6. No difference was observed in granzyme B, and CD3(-) CD127(+) cells were more abundant in native intestines. Ex vivo, and after in vitro activation, CD3(-) IELs in grafts produced significantly more interferon (IFN)-γ and IL-22, and a double IFNγ(+) IL-22(+) population was observed. Epithelial cell-depleted grafts IELs were cytotoxic, whereas this was not observed in controls. In conclusion, different from native intestines, a CD3(-) IEL subset predominates in grafts, showing features of natural killer cells and intraepithelial ILC1 (CD56(+) , NKp44(+) , CCR6(+) , CD127(-) , cytotoxicity, and IFNγ secretion), ILC3 (CD56(+) , NKp44(+) , IL-23R(+) , CCR6(+) , RORγt(+) , and IL-22 secretion), and intermediate ILC1-ILC3 phenotypes (IFNγ(+) IL-22(+) ). Viability of intestinal grafts may depend on the balance among proinflammatory and homeostatic roles of ILC subsets.

Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in Escherichia coli: comparison of in vivo with in vitro exposure of the supF gene.

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the supF gene of Escherichia coli were recovered from bacteria deficient in nucleotide excision repair and in DNA-alkyltransferase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7-fold above the spontaneous value. A total of 73 independent supF- mutants were sequenced. The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle-vector approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C-->A:T transitions. A substantial proportion (6/18) of CCNU-induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage-misalignment process in the generation of this mutation class. Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C-->A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O6-chloroethylguanine lesion with thymine. Most G:C-->A:T transitions (34/39) were located at three 5'-GG-3' hotspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C-->A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF-bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O6-chloroethylguanine formation, reducing its incidence in the transcribed DNA strand.

A single-blind, placebo-controlled study of glycosaminoglycan-peptide complex ('Rumalon') in patients with osteoarthritis of the hip or knee.

A randomized, single-blind, placebo-controlled trial was carried out in 62 patients (30 with osteoarthritis of the hip, 32 with osteoarthritis of the knee) to examine the efficacy of glycosaminoglycan-peptide complex in the treatment of osteoarthritis. Patients received 8-week courses of trial medication, each consisting of intramuscular injections of 3 x 2 ml ampoules per week, alternating with 8-week periods free of trial medication, in addition to conventional drug therapy and physiotherapy, as required. After 2-years' treatment, glycosaminoglycan-peptide-treated patients showed significant improvements, as compared with placebo, in relation to night pain, pain during the day, joint mobility and walking ability. Similar results were seen with both osteoarthritis of the hip and knee. In osteoarthritis of the knee it was also possible to assess joint swelling and this also showed a significant improvement. There were no significant changes in range of joint movement except for a significant decrease in active flexion in the patients with osteoarthritis of the knee treated with placebo. In contrast with many anti-osteoarthritic drugs, glycosaminoglycan-peptide complex was very well tolerated. These results suggest that glycosaminoglycan-peptide complex may be a valuable alternative form of long-term therapy for patients with osteoarthritis.

The effectiveness of the O(6)-alkylguanine-DNA alkyltransferase encoded by the ogt(ST) gene from S. typhimurium in protection against alkylating drugs, resistance to O(6)-benzylguanine and sensitisation to dibromoalkane genotoxicity.

Here we demonstrate that the Ogt(ST) from Salmonella typhimurium is a highly efficient O(6)-alkylguanine-DNA alkyltransferase (AGT) in affording protection against antitumour chloroethylating drugs (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)). In addition, Ogt(ST) is refractory to O(6)-benzylguanine (BG) inactivation and its expression provides only minor sensitisation to genotoxicity by environmental dibromoalkanes (DBE). No other of the assayed bacterial or human AGTs displayed such advantageous properties for chemoprotective gene therapy strategy. Our observations indicate that the Ogt(ST) AGT might be, under some circumstances, of potential use to improve cancer chemotherapy. At least, its properties may provide further insight into the design of human AGT variants that could be expressed in normal or tumour cells to provide either protection or ablation.

Quantitative relationship between mutagenic potency in the Ara test of Salmonella typhimurium and carcinogenic potency in rodents. A study of 11 direct-acting monofunctional alkylating agents.

This work attempted to derive a quantitative relationship between mutagenicity and carcinogenicity by examining the association between mutagenic potency in the Ara test of Salmonella typhimurium and carcinogenic potency in rodents. Mutagenesis was monitored by selecting forward mutations to L-arabinose resistance. Lethality was measured at equivalent experimental conditions to those of mutant yield by using a mixed population of a pair of isogenic strains distinguished by their differential nutritional requirements. The study was carried out with a group of 11 direct-acting monofunctional alkylating agents, which failed to show any quantitative correlation in the histidine reverse-mutation test. Our data suggest that the mutagenic efficiency of the compounds is directly proportional to the magnitude of the maximum yield of L-arabinose resistance mutants and inversely proportional to the dose and the number of lethal hits at which the maximum yield occurs. A highly significant correlation (r10 = 0.86, P less than 0.01) was found between the mutagenic efficiencies of the compounds in the Ara test and their carcinogenic potencies in rodents, expressed as TD50 ('tumor dose' 50) values. The result suggests that the Ara forward-mutation test of S. typhimurium might be capable of reflecting the relative potency of animal carcinogens, at least when confined to particular chemical classes. A more generic and definitive conclusion about the predictive value of the Ara test would require this analysis to be extended to other types of genotoxic carcinogens.

Influence of DNA repair by ada and ogt alkyltransferases on the mutational specificity of alkylating agents.

We investigated the influence of the alkyltransferases (ATases) encoded by the ada and ogt genes of Escherichia coli on the mutational specificity of alkylating agents. A new mutational assay for selection of supF- mutations in shuttle-vector plasmids was used. Treating plasmid-bearing bacteria with N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and ethyl methanesulfonate (EMS) dramatically increased the mutation frequency (from 33-fold to 789-fold). The vast majority of mutations (89-100%) were G:C-->A:T transitions. This type of mutation increased in ada- (MNU) or ogt- (ENU) bacteria, suggesting that repair of O6-methylguanine by ada ATase and repair of O6-ethylguanine by ogt ATase contribute mainly to the decrease in G:C-->A:T transitions. The analysis of neighboring base sequences revealed an overabundance of G:C-->A:T transitions at 5'-GG sequences. The 5'-PuG bias increased in ATase-defective cells, suggesting that these sequences were not refractory to repair. G:C-->A:T transitions occurred preferentially in the untranscribed strand after in vivo exposure. That this strand specificity was detected even in bacteria devoid of ATase activity (ada- ogt-) and not after in vitro mutagenesis suggests a bias for damage induction rather than for DNA repair. Highly significant differences were found between the in vivo and in vitro incidences of G:C-->A:T substitutions at the two major hotspots, positions 123 (5'-GGG-3'; antisense strand) and 168 (5'-GGA-3'; sense strand). These results are explained by differences in the probability of formation of stem-loop structures in vivo and in vitro.

Mutation spectra analysis suggests that N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli.

To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr-ada+ogt+, and uvr-ada-ogt-). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC-->AT transitions (the mutated G being almost exclusively preceded 5' by a purine). Mutation distribution was not random. Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt(+)-specific hot-spot. Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains. Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr- strains, whereas in the uvr- background, no difference was observed between mutation spectra in ada+ogt+ versus ada-ogt- strains. Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC-->AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli.

Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes.

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6-alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector-transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane-derived species to alkylate Trx.

ogt alkyltransferase enhances dibromoalkane mutagenicity in excision repair-deficient Escherichia coli K-12.

We examined the role of the O6-alkylguanine-DNA alkyltransferase encoded by ogt gene in the sensitivity of Escherichia coli to the mutagenic effects of the dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt- bacteria to those in their isogenic ogt+ parental counterparts. The effects of the uvrABC excision-repair system, the adaptive response, mucAB and umuDC mutagenic processing, and glutathione bioactivation on the differential responses of ogt- and ogt+ bacteria were also studied. Mutation induction was monitored by measuring the frequency of forward mutations to L-arabinose resistance. Induced mutations occurred only in excision repair-defective strains and were totally (with dibromomethane) or substantially (with dibromoethane) dependent on the alkyltransferase (ATase) encoded by the ogt gene. An increased mutagenic response to both dibromoalkanes was also seen in ogt- bacteria that overexpressed the ogt protein from a multicopy plasmid, indicating that the differences in mutability between ogt+ and ogt- bacteria were not dependent on the ogt- null allele carried by the defective strain. The ATase encoded by the constitutive ogt gene was more effective in promoting dibromoalkane mutagenicity than the ada ATase induced by exposure to low doses of a methylating agent. The mutagenicity promoted by the ogt ATase was dependent on both glutathione bioactivation and SOS mutagenic processing. To our knowledge, this paper presents for the first time evidence that DNA ATases, in particular the ATase encoded by the ogt gene, can increase the mutagenic effects of a DNA-damaging agent. The mechanism of this effect has yet to be established.

An association between mutagenicity of the Ara test of Salmonella typhimurium and carcinogenicity in rodents for 16 halogenated aliphatic hydrocarbons.

Sixteen halogenated aliphatic hydrocarbons were assayed for genotoxicity using the Ara mutagenicity assay with Salmonella typhimurium. Seven substances (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide, hexachloro-1,3-butadiene, iodoform and vinilydene chloride) were mutagenic at non-lethal doses. Comparatively, nine compounds (chloroform, carbon tetrachloride, 1,1,1-trichloroethane, 1,1,2-trichloroethane, tetrachloroethylene, trichloroethylene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane and hexachloroethane) were non-mutagenic after being assayed both in the presence and absence of metabolic activation with a rat liver microsomal fraction (S9). All negative compounds (except hexachloroethane) gave a lethal response, which could be an indication that bacteria were adequately exposed. The concordance between mutagenicity in the Ara test and carcinogenicity in rodents for this group of halogenated hydrocarbons was (31%) significantly lower than the concordance (72%) previously found in the Ara test with respect to a wider range of chemical classes. This result is in agreement with data reported for other genotoxicity assays. The presence of non-genotoxic carcinogens versus genotoxic non-carcinogens is discussed as a possible explanation. Five positive compounds (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide and hexachloro-1,3-butadiene) were analyzed for a quantitative relationship between carcinogenic potency in rats and the potency of response in the Ara mutagenicity test. This was possible because the Ara test, for volatile compounds (such as vinyl bromide), did not require the use of special vaporization techniques, which are difficult to evaluate quantitatively for mutagenic activity. A highly significant correlation was found between the mutagenic efficiencies of the five compounds in the Ara test and their carcinogenic potencies in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Human O(6)-alkylguanine-DNA alkyltransferase: protection against alkylating agents and sensitization to dibromoalkanes.

O(6)-alkylguanine-DNA alkyltransferase (AGT) is a suicide protein that corrects DNA damage by alkylating agents and may also serve to activate environmental carcinogens. We expressed human wild-type and two active mutant AGTs in bacteria that lack endogenous AGT and are also defective in nucleotide excision repair, to examine the ability of the AGTs to protect Escherichia coli from DNA damage by different types of alkylating agents and, oppositely, to sensitize cells to the genotoxic effects of dibromoalkanes (DBAs). Control bacteria carrying the cloning vector alone were extremely sensitive to mutagenesis by low, noncytotoxic doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Expression of human wild-type AGT prevented most of this enlarged susceptibility to MNNG mutagenesis. Oppositely, cell killing required much higher MNNG concentrations and prevention by wild-type AGT was much less effective. Mutants V139F and V139F/P140R/L142M protected bacteria against MNNG-induced cytotoxicity more effectively than the wild-type AGT, but protection against the less stringent mutagenesis assay was variable. Subtle differences between wild-type AGT and the two mutant variants were further revealed by assaying protection against mutagenesis by more complex alkylating agents, such as N-ethyl-N-nitrosourea and 1-(2-chloro- ethyl)-3-cyclohexyl-1-nitrosourea. Unlike wild-type and V139F, the triple mutant variant, V139F/P140R/L142M was unaffected by the AGT inhibitor, O(6)-benzylguanine. Wild-type AGT and V139F potentiated the genotoxic effects of DBAs; however, the triple mutant virtually failed to sensitize the bacteria to these agents. These experiments provide evidence that in addition to the active site cysteine at position 145, the proline at position 140 might be important in defining the capacity by which AGTs modulate genotoxicity by environmentally relevant DBAs. The ability of AGTs to activate dibromoalkanes suggests that this DNA repair enzyme could be altered, and if expressed in tumors might be lethal by enhancing the activation of specific chemotherapeutic prodrugs.