Genetic polymorphism in Methylenetetrahydrofolate Reductase chloride transport protein 6 (MTHFR CLCN6) gene is associated with keratinocyte skin cancer in a cohort of renal transplant recipients.

Background: Renal transplant recipients (RTRs) are at increased risk of keratinocyte cancer (KC), especially cutaneous squamous cell carcinoma (cSCC). Previous studies identified a genetic variant of the Methylenetetrahydrofolate Reductase (MTHFR) gene, C677T, which conferred a risk for diagnosis of cSCC in Irish RTRs. Objective: We sought to find further genetic variation in MTHFR and overlap genes that may be associated with a diagnosis of KC in RTRs. Methods: Genotyping of a combined RTR population (n = 821) from two centres, Ireland (n = 546) and the USA (n = 275), was performed. This included 290 RTRs with KC and 444 without. Eleven single nucleotide polymorphisms (SNPs) in the MTHFR gene and seven in the overlap gene MTHFR Chloride transport protein 6 (CLCN6) were evaluated and association explored by time to event analysis (from transplant to first KC) using Cox proportional hazards model. Results: Polymorphism at MTHFR CLCN6 (rs9651118) was significantly associated with KC in RTRs (HR 1.50, 95% CI 1.17-1.91, p < 0.00061) and cSCC (HR 1.63, 95% CI 1.14-2.34, p = 0.007). A separate SNP, MTHFR C677T, was also significantly associated with KC in the Irish population (HR 1.31, 95% CI 1.05-1.63, p = 0.016), but not American RTRs. Conclusions: We report the association of a SNP in the MTHFR overlap gene, CLCN6 and KC in a combined RTR population. While the exact function of CLCN6 is not known, it is proposed to be involved in folate availability. Future applications could include incorporation in a polygenic risk score for KC in RTRs to help identify those at increased risk beyond traditional risk factor assessment.

α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFß antibody to promote durable rejection and immunity in squamous cell carcinomas.

BACKGROUND: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFß signaling. METHODS: To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFß or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. RESULTS: We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFß monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFß, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFß antibody administration attenuates these effects. We show that α-TGFß acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFß acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. CONCLUSIONS: We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFß-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFß combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFß/α-PD-1 combination therapy (NCT02947165).

The requirement for early exposure of Haemonchus contortus larvae to Bacillus thuringiensis for effective inhibition of larval development.

The potential for a nematocidal Bacillus thuringiensis (Bt) to target the free-living larval stages of Haemonchus contortus was examined using in vitro larval development and migration assays. Bt toxicity in larval development assays decreased as the time period between egg hatch and initial exposure to the Bt was increased; a time lag of 48 h resulted in a 350-fold increase in the IC(50) (from 2.6 ng/ml to 910 ng/ml). The effects on larval migration largely paralleled the effects on larval development, indicating that the larvae which reached the infective stage after exposure to Bt were generally as 'fit' as control worms in terms of migration ability. However, a comparison of the two assays also showed the presence of a level of Bt exposure which showed significantly more toxicity in migration assays than development assays, indicating that, in some cases, fully developed Bt-exposed larvae were less able to migrate than controls, and hence may be compromised in their ability to infect sheep. The rapid decrease in toxicity when exposure to the Bt is delayed highlights a significant issue concerning the use of Bt for control of the free-living larval stages of animal-parasitic nematodes. Targeting the larvae by delivering bacterial spores to the faeces through the host animal's digestive tract would require the spores to germinate upon defecation, grow through a vegetative phase, to then produce crystal toxin protein upon subsequent sporulation. This period of bacterial development will introduce a time lag between worm egg hatching and initial exposure of the larvae to the Bt, which, as demonstrated in the present study, may allow the worm larvae to develop to late larval stages which are relatively insensitive to the toxin.

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria.

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.

Toxicity of Bacillus thuringiensis to parasitic and free-living life-stages of nematode parasites of livestock.

A collection of Bacillus thuringiensis (Bt) strains (Bts) were screened for activity against the free-living larval stages of nematode parasites of livestock. Two strains were identified with significant activity in inhibiting larval development of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta. These strains were also toxic to the adult parasitic stages of these nematode species in vitro. Adult H. contortus and O. circumcincta showed complete cessation of movement within 2 and 4 days, respectively. Trichostrongylus colubriformis adults were less affected, however, movement was still significantly reduced compared with controls. The in vitro activity against the larval stages was of a magnitude similar to or greater than that seen with the anthelmintic drugs thiabendazole and levamisole. N-terminal amino acid sequencing indicated that the two Bts contained either Cry5A and Cry5B proteins, or a Cry13 protein, and the presence of the corresponding cry5A, cry5B and cry13 genes was confirmed by PCR and sequencing. Bacillus thuringiensis spore-crystal suspensions exposed to acidic pH conditions (pH

Taxonomy of Australian clinical isolates of the genus Photorhabdus and proposal of Photorhabdus asymbiotica subsp. asymbiotica subsp. nov. and P. asymbiotica subsp. australis subsp. nov.

The relationship of Photorhabdus isolates that were cultured from human clinical specimens in Australia to Photorhabdus asymbiotica isolates from human clinical specimens in the USA and to species of the genus Photorhabdus that are associated symbiotically with entomopathogenic nematodes was evaluated. A polyphasic approach that involved DNA-DNA hybridization, phylogenetic analyses of 16S rRNA and gyrB gene sequences and phenotypic characterization was adopted. These investigations showed that gyrB gene sequence data correlated well with DNA-DNA hybridization and phenotypic data, but that 16S rRNA gene sequence data were not suitable for defining species within the genus Photorhabdus. Australian clinical isolates proved to be related most closely to clinical isolates from the USA, but the two groups were distinct. A novel subspecies, Photorhabdus asymbiotica subsp. australis subsp. nov. (type strain, 9802892T=CIP 108025T=ACM 5210T), is proposed, with the concomitant creation of Photorhabdus asymbiotica subsp. asymbiotica subsp. nov. Analysis of gyrB sequences, coupled with previously published data on DNA-DNA hybridization and PCR-RFLP analysis of the 16S rRNA gene, indicated that there are more than the three subspecies of Photorhabdus luminescens that have been described and confirmed the validity of the previously proposed subdivision of Photorhabdus temperata. Although a non-luminescent, symbiotic isolate clustered consistently with P. asymbiotica in gyrB phylogenetic analyses, DNA-DNA hybridization indicated that this isolate does not belong to the species P. asymbiotica and that there is a clear distinction between symbiotic and clinical species of Photorhabdus.

Factors affecting the toxicity of Bacillus thuringiensis var. israelensis and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi (Diptera: Chironomidae).

Laboratory bioassays (48h duration, 25+/-1 degrees C) were used to determine the toxicity of Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi, a major pest of rice in southern Australia. Bioassays were conducted using different combinations of larval ages and densities to determine if these factors affected toxicity. The effects of temperature and substrate type on B.t.i. toxicity were also investigated. Tests were conducted using a commercial B.t.i. formulation (VectoBac WDG, 3000ITU/mg), a spore/crystal mixture derived from the VectoBac WDG strain, and VectoLex WDG, a commercial B. sphaericus formulation (650ITU/mg). VectoBac WDG was highly toxic to fourth instar C. tepperi in bioassays using a sand substrate (LC(50) 0.46mg/L, older larvae); younger fourth instar larvae were more susceptible (LC(50) 0.20mg/L). Increasing larval densities (from 10 to 30 per bioassay cup) increased LC(50) values for both age groups, significantly so in the case of older larvae (higher density LC(50) 0.80mg/L). Use of a soil substrate increased the LC(50) value (older larvae, 10 per cup) to 0.99mg/L. Similar differences in toxicity relative to larval age and substrate type were found in bioassays using the B.t.i. spore/crystal mixture. VectoBac WDG and the spore/crystal mixture both showed similar (approximately 6-fold) declines in activity between 30 and 17.5 degrees C. At lower temperatures (between 17.5 and 15 degrees C), activity of the spore/crystal mixture declined much more rapidly than that of VectoBac WDG. VectoLex WDG showed very low toxicity to C. tepperi larvae, and the overall impact of larval age and density was relatively minor (LC(50) values 1062-1340mg/L). Autoclaving VectoLex WDG did not substantially reduce its toxicity (LC(50) 1426mg/L), suggesting that formulation additives (i.e., surfactants and other adjuvants) are responsible for much of the toxicity occurring at the high product concentrations required to cause C. tepperi mortality. Whilst VectoLex WDG was ineffective against C. tepperi, VectoBac WDG has the potential to provide selective control of this rice pest at economically viable application rates.

Antimicrobial resistance and genomic screening of clinical isolates of thermophilic Campylobacter spp. from south-east Queensland, Australia.

AIM: To screen 90 clinical isolates of thermophilic Campylobacter species for putative resistance to ampicillin, erythromycin and tetracycline and perform numerical analysis to determine isolate relatedness. METHODS AND RESULTS: Disc diffusion, E-test MIC and agar dilution methods were performed. Disc diffusion testing showed 87 (97%) isolates appeared resistant to ampicillin at 10 microg; 14 (16%) resistant to tetracycline at 30 microg; and three (3.4%) resistant to erythromycin at 15 microg. E-test MICs showed a range of 0.5 to >256 mg l(-1) for ampicillin; 16 to >256 mg l(-1) for tetracycline; and >256 mg l(-1) for erythromycin. E-test showed 68% correlation (+/-1 log2 dilution) with agar dilution for ampicillin, 100% for erythromycin and 64% for tetracycline. Disc diffusion testing showed 100% correlation with agar dilution for erythromycin and tetracycline, and 77% for ampicillin. Numerical analyses of restriction endonuclease (RE) fragment profiles suggested a high level of isolate variation. CONCLUSION: The incidence of resistance of thermophilic Campylobacter spp. to erythromycin and tetracycline is low in south-east Queensland. SIGNIFICANCE AND IMPACT OF THE STUDY: Disc diffusion susceptibility testing may be used to screen thermophilic Campylobacter spp. for putative resistance to erythromycin and tetracycline. Agar dilution should be used to determine ampicillin susceptibility.

TGF-beta signaling in cancer--a double-edged sword.

Transforming growth factor (TGF) beta1 is a potent growth inhibitor, with tumor-suppressing activity. Cancers are often refractile to this growth inhibition either because of genetic loss of TGF-beta signaling components or, more commonly, because of downstream perturbation of the signaling pathway, such as by Ras activation. Carcinomas often secrete excess TGF-beta1 and respond to it by enhanced invasion and metastasis. Therapeutic approaches should aim to inhibit the TGF-beta-induced invasive phenotype, but also to retain its growth-inhibitory and apoptosis-inducing effects.

TGF-beta signaling in tumor suppression and cancer progression.

Epithelial and hematopoietic cells have a high turnover and their progenitor cells divide continuously, making them prime targets for genetic and epigenetic changes that lead to cell transformation and tumorigenesis. The consequent changes in cell behavior and responsiveness result not only from genetic alterations such as activation of oncogenes or inactivation of tumor suppressor genes, but also from altered production of, or responsiveness to, stimulatory or inhibitory growth and differentiation factors. Among these, transforming growth factor beta (TGF-beta) and its signaling effectors act as key determinants of carcinoma cell behavior. The autocrine and paracrine effects of TGF-beta on tumor cells and the tumor micro-environment exert both positive and negative influences on cancer development. Accordingly, the TGF-beta signaling pathway has been considered as both a tumor suppressor pathway and a promoter of tumor progression and invasion. Here we evaluate the role of TGF-beta in tumor development and attempt to reconcile the positive and negative effects of TGF-beta in carcinogenesis.

Screening for novel cry genes by hybridization.

AIMS: To develop an efficient and sensitive method to facilitate the search for novel Cry toxins. METHODS AND RESULTS: The method uses a cocktail of cry gene sequences as a hybridization probe to screen Bacillus thuringiensis (Bt) strains and gene libraries prepared from them. Under the hybridization and washing conditions used, cross-hybridization between genes from different cry families was not observed. Probes containing either partial or complete cry gene sequences produced similar patterns when hybridized to genomic DNA of several Bt strains although the pattern produced by the probe composed of entire gene coding regions was somewhat more complex. CONCLUSION: As a tool for gene library screening, hybridization with a mixture of cry gene sequences is an efficient means of detecting clones containing a diverse range of cry genes in a single step. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique greatly improves the ease and efficiency of novel toxin gene discovery compared to previous methods.

Isolation, identification, and molecular characterization of strains of Photorhabdus luminescens from infected humans in Australia.

We describe the isolation of Photorhabdus (Xenorhabdus) luminescens from four Australian patients: two with multiple skin lesions, one with bacteremia only, and one with disseminated infection. One of the patients had multiple skin lesions following the bite of a spider, while the lesions in the other patient were possibly associated with a spider bite. The source of infection for the remaining two patients is unknown. As a member of the family Enterobacteriaceae, P. luminescens is unusual in that it fails to reduce nitrate and ferments only glucose and mannose. It gives negative reactions for lysine decarboxylase, arginine dihydrolase, and ornithine decarboxylase (Moeller). The species is motile, utilizes citrate, hydrolyzes urea, and usually produces a unique type of annular hemolysis on sheep blood agar plates incubated at 25 degrees C. A weak bioluminescence is the defining characteristic. P. luminescens is an insect pathogen and is symbiotically associated with entomopathogenic nematodes. Its isolation from human clinical specimens has been reported previously from the United States. Restriction fragment length polymorphism-PCR analysis of the 16S rRNA gene demonstrated a high level of similarity among the Australian clinical strains and significant differences between the Australian clinical strains and the U.S. clinical strains. However, numerical analyses of the data suggest that the two groups of clinical strains are more similar to each other than they are to the symbiotic strains found in nematodes. This is the first report of the isolation of P. luminescens from infected humans in Australia and the second report of the isolation of this species from infected humans worldwide.

Genetic events and the role of TGF beta in epithelial tumour progression.

The mouse skin model of chemical carcinogenesis has been very well characterized with respect to epigenetic changes, which occur during tumour cell initiation, promotion and progression. The use of transgenic and gene knock-out mice has contributed greatly to knowledge in this area. The H-ras genetic locus has been shown to undergo multiple genetic changes, including mutagenic activation, amplification of the mutant gene, and loss of the normal allele. These different genetic events lead to thresholds of ras activity which contribute to different stages along the pathway to neoplasia. The genetic and epigenetic events which lead to tumour invasion and metastasis have been less well characterized than studies on tumour initiation and promotion, despite the fact that it is metastases which ultimately kill the animal/patient. In the mouse skin model, loss of p53 contributes to malignant conversion. Gene deletion of the INK4 locus is associated with transformation to a highly invasive spindle cell tumor phenotype. This spindle cell transformation can also be induced in vitro or in vivo by TGF beta 1, possible by synergizing with mutant H-ras. TGF beta can have both positive and negative effects on tumourigenesis, acting early as a tumour suppresser, but later as a stimulator of tumour invasion. It is this latter effect which may be clinically more significant, since many human tumours overexpress TGF beta, yet the majority still retain the intracellular signaling systems necessary for the cell to respond to this growth factor.

Transforming growth factor beta is essential for spindle cell conversion of mouse skin carcinoma in vivo: implications for tumor invasion.

Transforming growth factor beta1 (TGF-beta1) regulates both cell growth and cellular plasticity and is therefore important in the molecular control of both the developmental and neoplastic processes. It has been suggested that TGF-beta1 may be a positive or negative regulator of tumorigenesis. Stimulation of tumorigenesis could be due to its action as an immunosuppressor or as an inducer of angiogenesis, or by its direct action on the cell in promoting cellular plasticity. In the current study, we provide evidence that TGF-beta1 can act directly on keratinocytes in vivo to induce the reversible epithelial-mesenchymal conversion of a malignant metastatic keratinocyte cell line. Two squamous clones from the cell line were shown to undergo a reversible conversion to a fibroblastoid phenotype after culture in 1 ng/ml TGF-beta1. The morphological conversion became apparent at 24 h post-TGF-beta treatment and was complete after another 24 h. The conversion was characterized by a rapid delocalization of E-cadherin within 6-12 h posttreatment, followed by down-regulation of E-cadherin levels by 72 h. These squamous clones spontaneously converted to a fibroblastoid phenotype after s.c. injection in nude mice. Importantly, four of four clones that had been stably transfected with a dominant negative TGF-beta type II receptor were unable to undergo this mesenchymal switch in vivo, despite the fact that all clones stably transfected with neomyocin resistance alone retained their spindle characteristics in vivo. This demonstrates that the epithelial-mesenchymal conversion event is mediated directly via the TGF-beta signaling pathway of the tumor cell per se, and that it is sufficient to significantly enhance tumorigenicity and the malignant and invasive characteristics of the tumor in vivo.

Fast and accurate identification of Xenorhabdus and Photorhabdus species by restriction analysis of PCR-amplified 16S rRNA genes.

Thirteen bacterial strains of Xenorhabdus and 14 strains of Photorhabdus originating from a wide range of geographical and nematode host sources were typed by analyzing 16S rRNA gene (rDNA) restriction patterns obtained after digestion of PCR-amplified 16S rDNAs. Eight tetrameric restriction endonucleases were examined. A total of 17 genotypes were identified, forming two heterogeneous main clusters after analysis by the unweighted pair-group method using arithmetic averages: group I included all Xenorhabdus species and strains, symbionts of Steinernema, whereas group II encompassed the Photorhabdus strains, symbionts of Heterorhabditis. To identify the four valid species of Xenorhabdus and unclassified strains and all the genotypes of Photorhabdus luminescens, three restriction enzymes are required: CfoI, AluI, and HaeIII. Our results, in substantial agreement with DNA-DNA pairing and 16S rDNA sequence data, indicate that amplified 16S rDNA restriction analysis is a simple and accurate tool for identifying entomopathogenic nematode bacterial symbionts.

Mapping of a major genetic modifier of embryonic lethality in TGF beta 1 knockout mice.

The transforming growth factor beta 1 (TGF beta 1) signalling pathway is important in embryogenesis and has been implicated in hereditary haemorrhagic telangiectasia (HHT), atherosclerosis, tumorigenesis and immunomodulation. Therefore, identification of factors which modulate TGF beta 1 bioactivity in vivo is important. On a mixed genetic background, approximately 50% Tgfb1-/- conceptuses die midgestation from defective yolk sac vasculogenesis. The other half are developmentally normal but die three weeks postpartum. Intriguingly, the vascular defects of Tgfb1-/- mice share histological similarities to lesions seen in HHT patients. It has been suggested that dichotomy in Tgfb1-/- lethal phenotypes is due to maternal TGF beta 1 rescue of some, but not all, Tgfb1-/- embryos12. Here we show that the Tgfb1-/- phenotype depends on the genetic background of the conceptus. In NIH/Ola, C57BL/6J/Ola and F1 conceptuses, Tgfb1-/- lethality can be categorized into three developmental classes. A major codominant modifier gene of embryo lethality was mapped to proximal mouse chromosome 5, using a genome scan for non-mendelian distribution of alleles in Tgfb1-/- neonatal animals which survive prenatal lethality. This gene accounts for around three quarters of the genetic effect between mouse strains and can, in part, explain the distribution of the three lethal phenotypes. This approach, using neonatal DNA samples, is generally applicable to identification of loci that influence the effect of early embryonic lethal mutations, thus furthering knowledge of genetic interactions that occur during early mammalian development in vivo.

A 16S rRNA gene oligonucleotide probe for identification of Bacillus thuringiensis isolates from sheep fleece.

Larvae of the Australian sheep blowfly Lucilia cuprina are susceptible to some strains of Bacillus thuringiensis, including some isolates from sheep fleeces from South Australia and Western Australia. Larvicidal strains of B. thuringiensis include var. thuringiensis, tolworthi, darmstadiensis, morrisoni, and toumanoffi. As part of an ecological study, a large number of B. thuringiensis fleece isolates from untreated sheep and sheep previously treated with larvicidal B. thuringiensis were screened for larvicidal activity. A ribotyping method to distinguish larvicidal B. thuringiensis jetted (sprayed) onto sheep from the natural B. thuringiensis flora of the fleece was developed. A cloned fragment of the 16S rRNA gene used as a probe for B. thuringiensis DNA digested with restriction endonucleases enables separation of B. thuringiensis serovars and separation within the serovars thuringiensis, tolworthi, and kurstaki. The 16S gene probe method is useful for distinguishing between reisolates of larvicidal B. thuringiensis jetted onto sheep and the background B. thuringiensis population present prior to jetting.

Phenotypic and DNA relatedness between nematode symbionts and clinical strains of the genus Photorhabdus (Enterobacteriaceae).

Bacterial strains isolated from wide ranges of nematode hosts and geographic sources and strains isolated from human clinical specimens were used to assess the taxonomic structure of the genus Photorhabdus. The following two methods were used: DNA relatedness and phenotypic characterization. Analysis of the DNA relatedness data revealed that all of the strains studied were congeneric and that the genus Photorhabdus is, on the basis of DNA relatedness data, more homogeneous than the other genus of nematode-symbiotic bacteria, the genus Xenorhabdus. In contrast to previous reports, only two DNA relatedness groups were identified in the genus Photorhabdus. These groups corresponded to the symbiotic strains and the clinical strains. There appeared to be some subgroups within the symbiotic strain group on the basis of the interactions of the strains with nematodes, which corresponded to some extent with the DNA relatedness data. However, there were significant ambiguities in the DNA relatedness data, and this group could not be subdivided on the basis of DNA relatedness data or phenotypic data. The distinct functional differences within and between the DNA relatedness groups of symbiotic Photorhabdus strains indicated that there are biologically significant sub-groups within the genus Photorhabdus that cannot be defined at this time. Further investigation of the taxonomy of Photorhabdus by using different approaches and a suitably wide range of strains is recommended. However, it is clear that the clinical strains form a recognizable subgroup within the genus even though no formal subtaxon can be defined at this time.

TGFbeta1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice.

TGFbeta1 has been implicated in cell cycle control and carcinogenesis. To address the exact function of TGFbeta1 in skin carcinogenesis in vivo, mice with TGFbeta1 expression targeted to keratinocytes were subjected to long-term chemical carcinogenesis treatment. TGFbeta1 showed biphasic action during multistage skin carcinogenesis, acting early as a tumor suppressor but later enhancing the malignant phenotype. The transgenics were more resistant to induction of benign skin tumors than controls, but the malignant conversion rate was vastly increased. There was also a higher incidence of spindle cell carcinomas, which expressed high levels of endogenous TGFbeta3, suggesting that TGFbeta1 elicits an epithelial-mesenchymal transition in vivo and that TGFbeta3 might be involved in maintenance of the spindle cell phenotype. The action of TGFbeta1 in enhancing malignant progression may mimic its proposed function in modulating epithelial cell plasticity during embryonic development.

Altered epidermal cell growth control in vivo by inducible expression of transforming growth factor beta 1 in the skin of transgenic mice.

An inducible bovine KIV* keratin gene promoter was used to target expression of latent or activated transforming growth factor beta 1 (TGF beta 1) to keratinocytes in transgenic mice. This short (2.2-kb) keratin 6 (K6) promoter element was generally silent in untreated animals but was induced in keratinocytes when placed in culture or, in vivo, in response to hyperplasia that follows topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All of the K6-TGF beta 1 transgenic lines studied showed attenuation of the basal keratinocyte proliferative response to 12-O-tetradecanoylphorbol-13-acetate as a consequence of inducible TGF beta 1 gene expression. One of the six lines studied showed constitutive transgene expression at low levels in the skin, and this line had a 2- to 3-fold increase in epidermal DNA labeling index over control mice. Although in vitro TGF beta 1 is known to be a potent negative regulator of epithelial cell proliferation, in vivo TGF beta 1 has complex biological activities and can act as either a positive or negative regulator of keratinocyte proliferation.