Diphtheria toxoid dissolving microneedle vaccination: Adjuvant screening and effect of repeated-fractional dose administration.

In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20 min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5 h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.

Inflammation and wound healing in cats with chronic gingivitis/stomatitis after extraction of all premolars and molars were not affected by feeding of two diets with different omega-6/omega-3 polyunsaturated fatty acid ratios.

Feline chronic gingivitis/stomatitis (FCGS) is a painful inflammatory disease in cats. Extraction of teeth, including all premolars and molars, has been shown to be the therapy of choice in cats not responding sufficiently to home care (e.g. tooth brushing) and/or medical treatment (corticosteroids and/or antibiotics). In this study, we hypothesize that a cat food with an omega-6 polyunsaturated fatty acid (ω6 PUFA) to ω3 PUFA ratio of 10:1 reduces inflammation of the FCGS and accelerates soft tissue wound healing of the gingiva after dental extractions, compared to a cat food with a ω6:ω3 PUFA ratio of 40:1. The cats were fed diets with chicken fat and fish oil as sources of fatty acids. In one diet, part of the fish oil was replaced by safflower oil, resulting in two diets with ω6:ω3 PUFA ratios of 10:1 and 40:1. This double-blinded study in two groups of seven cats revealed that dietary fatty acids influence the composition of plasma cholesteryl esters and plasma levels of inflammatory cytokines. The diet with the 10:1 ratio lowered PGD(2) , PGE(2) and LTB(4) plasma levels significantly, compared to the diet with the 40:1 ratio (p = 0.05, p = 0.04, and p = 0.02 respectively). However, feeding diets with dietary ω6:ω3 PUFA ratios of 10:1 and 40:1, given to cats with FCGS for 4 weeks after extraction of all premolars and molars, did not alter the degree of inflammation or wound healing.

Osteoclast progenitors from cats with and without tooth resorption respond differently to 1,25-dihydroxyvitamin D and interleukin-6.

Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)(2)D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR(+) and TR(-)) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14 days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR(-) PBMCs than from TR(+) PBMCs on plastic. More osteoclasts were formed from TR(+) PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)(2)D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR(+) osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR(+) PBMCs when cultured on bone are sensitive to 1,25(OH)(2)D, whereas the differentiation of TR(-) PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors.

Comparison of periodontal pathogens between cats and their owners.

The periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia are strongly associated with periodontal disease and are highly prevalent in humans with periodontitis. Porphyromonas and Tannerella spp. have also been isolated from the oral cavity of cats. The oral microflora in animals was compared with those in humans in earlier studies, but no studies are available on the comparison of the oral microflora from pets and their respective owners. The aim of this study was to determine the presence of these bacteria in the oral microflora of cats and their owners, since animal to human transmission, or vice versa, of oral pathogens could have public health implications. This study investigated the prevalence of Porphyromonas gulae, P. gingivalis, and T. forsythia in the oral microflora of cats and their owners, using culture and polymerase chain reaction (PCR). All Porphyromonas isolates from cats (n=64) were catalase positive, whereas the Porphyromonas isolates from owners (n=7) were catalase negative, suggesting that the isolates from cats were P. gulae whereas those from the owners were P. gingivalis. T. forsythia was recovered from both cats (n=63) and owners (n=31); the proportion of T. forsythia relative to the total CFU was higher in cats with periodontitis than in cats without periodontal disease. Genotyping of T. forsythia isolates (n=54) in six cat/owner couples showed that in one cat/owner couple the T. forsythia isolates (n=6) were identical. These T. forsythia isolates were all catalase positive, which led us to hypothesize that transmission from cats to owners had occurred and that cats may be a reservoir of T. forsythia.

[Effectiveness of feeding large kibbles with mechanical cleaning properties in cats with gingivitis]. / Effectiviteit van het voeren van een grote brok met mechanisch reinigende werking bij katten met gingivitis.

Periodontal disease is the most common acquired oral disease in cats. It starts with plaque accumulation and gingivitis. The aim of this study was to evaluate the effectiveness of different types of kibble and teeth brushing after professional dental cleaning. To this end, the ratio of facultative anaerobic and obligate anaerobic bacteria was determined, and the amount of plaque and tartar and the severity of gingivitis were measured. Eighty-eight cats with gingivitis and tartar were randomly divided into four groups and given different treatments after professional dental cleaning. The dental treatments were: group 1, cats were fed on small kibbles with mechanical cleaning qualities; group 2, cats were fed on small kibbles without mechanical cleaning qualities, and owners were instructed to brush the cats' teeth; group 3, cats were fed on large kibbles with mechanical cleaning qualities; and group 4, cats were fed on the same kibbles as group 2, but owners were not asked to brush their cats' teeth. A supragingival plaque sample was taken, and used for bacteriological analysis scores for tartar, plaque, and gingivitis were determined at the initial presentation and at 3 and 6 months after professional dental cleaning. Cats fed on the large kibbles with mechanical cleaning qualities (group 3) had significantly less gingivitis and tartar.

Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay.

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.

DMBA-induced toxic and mutagenic responses vary dramatically between NER-deficient Xpa, Xpc and Csb mice.

Heterogeneity in cancer susceptibility exists between patients with an inherited defect in nucleotide excision repair (NER). While xeroderma pigmentosum (XP) patients have elevated skin cancer rates, Cockayne syndrome (CS) patients do not appear to have increased cancer susceptibility. To investigate whether differences in mutagenesis are the basis for the variability in cancer proneness, we studied mutagenesis at the X-chromosomal Hprt gene and the autosomal Aprt gene in splenic T-lymphocytes after 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure in total NER-deficient Xpa mice, global genome repair (GGR)-deficient Xpc mice and transcription coupled repair (TCR)-deficient Csb mice. Surprisingly, while all intraperitoneally-treated Xpc(-/-) mice survived a dose of 40 mg/kg DMBA, a substantial fraction of the treated Xpa(-/-) and Csb(-/-) mice died a few days after treatment with a 20-fold lower dose. Functional TCR of DMBA adducts in Xpc(-/-) mice thus appears to alleviate DMBA toxicity. However, the mutagenic response in Xpc(-/-) mice was +/- 2-fold enhanced at both the Hprt and the Aprt gene compared to heterozygous controls, indicating that GGR at least partially removes DMBA adducts from the genome overall. DMBA-induced SCE frequencies in mouse dermal fibroblasts were significantly enhanced in Xpa- and Csb-, but not in Xpc-deficient background compared to the frequency in normal fibroblasts. These results indicate that both damage-induced cytotoxicity as well as intra-chromosomal recombinational events were not correlated to differences in cancer susceptibility in human NER syndrome patients.

Gene and sequence specificity of DNA damage induction and repair: consequences for mutagenesis.

The field of DNA repair has been expanded enormously in the last 20 years. In this paper, work on gene and sequence specificity of DNA damage induction and repair is summarized in the light of the large and broad contribution of Phil Hanawalt to this field of research. Furthermore, the consequences of DNA damage and repair for mutation induction is discussed, and the contribution of Paul Lohman to the development of assays employing transgenic mice for the detection of gene mutations is highlighted.

Mouse mismatch repair gene Msh2 is not essential for transcription-coupled repair of UV-induced cyclobutane pyrimidine dimers.

The human mutS homolog gene MSH2 is essential for DNA mismatch repair (MMR) and defects in this gene can result in increased mutagenesis, genomic instability and hereditary nonpolyposis colorectal cancer (HNPCC). Besides correcting mismatch errors arising from DNA replication, it was shown that deficiencies in bacterial and human MMR genes including MSH2 resulted in defective transcription-coupled repair (TCR) of UV-induced photolesions. Here we show that MMR-deficient fibroblasts derived from two independent isogenic mouse strains with defined Msh2 deficiencies are as proficient in TCR of UV-induced cyclobutane pyrimidine dimers (CPD) as wildtype fibroblasts. Our results indicate that in mouse cells Msh2 is not essential for TCR of UV-induced CPD in contrast to bacteria and human cells and suggest that the biological effects of UV in mouse Msh2(-/-) cells and mice are not due to defective TCR.

Loss of heterozygosity in somatic cells of the mouse. An important step in cancer initiation?

Loss of heterozygosity (LOH) of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Recently, we and others have developed mouse models in which the frequency and nature of LOH events at an autosomal locus can be elucidated in genetically stable normal somatic cells. In this paper, an overview is presented of recent studies in LOH-detecting mouse models. Molecular mechanisms that lead to LOH and the effects of genetic and environmental variables are discussed. The general finding that LOH of a marker gene occurs frequently in somatic cells of the mouse without deleterious effects on cell viability, suggests that also tumour suppressor genes are lost in similar frequencies. LOH of tumour suppressor genes may thus be an initiating event in cancer development.

The relationship between benzo[a]pyrene-induced mutagenesis and carcinogenesis in repair-deficient Cockayne syndrome group B mice.

Cockayne syndrome (CS) patients are deficient in the transcription coupled repair (TCR) subpathway of nucleotide excision repair (NER) but in contrast to xeroderma pigmentosum patients, who have a defect in the global genome repair subpathway of NER, CS patients do not have an elevated cancer incidence. To determine to what extent a TCR deficiency affects carcinogen-induced mutagenesis and carcinogenesis, CS group B correcting gene (CSB)-deficient mice were treated with the genotoxic carcinogen benzo(a)pyrene (B[a]P) at an oral dose of 13 mg/kg body weight, three times a week. At different time points, mutant frequencies at the inactive lacZ gene (in spleen, liver, and lung) as well as at the active hypoxanthine phosphoribosyltransferase (Hprt) gene (in spleen) were determined to compare mutagenesis at inactive versus active genes. B[a]P treatment gave rise to increased mutant frequencies at lacZ in all of the organs tested without a significant difference between CSB-/- and wild-type mice, whereas B[a]P-induced Hprt mutant frequencies in splenic T-lymphocytes were significantly more enhanced in CSB-/- mice than in control mice. The sequence data obtained from Hprt mutants indicate that B[a]P adducts at guanine residues were preferentially removed from the transcribed strand of the Hprt gene in control mice but not in CSB-/- mice. On oral treatment with B[a]P, the tumor incidence increased in both wild-type and CSB-deficient animals. However, no differences in tumor rate were observed between TCR-deficient CSB-/- mice and wild-type mice, which is in line with the normal cancer susceptibility of CS patients. The mutagenic response at lacZ, in contrast to Hprt, correlated well with the cancer incidence in CSB-/- mice after B[a]P treatment, which suggests that mutations in the bulk of the DNA (inactive genes) are a better predictive marker for carcinogen-induced tumorigenesis than mutations in genes that are actively transcribed. Thus, the global genome repair pathway of NER appears to play an important role in the prevention of cancer.

Age-dependent spontaneous mutagenesis in Xpc mice defective in nucleotide excision repair.

DNA damages caused by cellular metabolites and environmental agents induce mutations, that may predispose to cancer. Nucleotide excision repair (NER) is a major cellular defence mechanism acting on a variety of DNA lesions. Here, we show that spontaneous mutant frequencies at the Hprt gene increased 30-fold in T-lymphocytes of 1 year old Xpc-/- mice, possessing only functional transcription-coupled repair (TCR). Hprt mutant frequencies in Xpa-/- and Csb-/- mice that both have a defect in this NER subpathway, remained low during ageing. In contrast to current models, the elevated mutation rate in Xpc-/- mice does not lead to an increased tumour incidence or premature ageing. Oncogene (2000) 19, 5034 - 5037

UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II.

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged beta-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.

Heterozygous Aprt mouse model: detection and study of a broad range of autosomal somatic mutations in vivo.

During the development of cancer a series of specific genetic alterations have to occur in a stepwise fashion to transform a normal somatic cell into a malignant tumor cell. These genetic changes can be roughly divided in two groups: mutations in proto-oncogenes that result in a constantly activated gene product and mutations in tumor-suppressor genes that result in loss of function. While oncogenic mutations often have a dominant phenotype and mutation of one allele is sufficient for activation, in general both alleles of a tumor suppressor gene have to be disrupted to abolish its function. The requested specificity for activating mutations in proto-oncogenes is high, since only a limited number of mutations at specific sites result in an activated protein. In contrast, disruption of a tumor suppressor gene can be accomplished via various mechanisms. Familial cancers often contain a germline mutation in one allele of a tumor suppressor gene. In tumors, the second allele is then frequently lost by genetic alterations that also affect the heterozygous state of multiple loci adjacent to the tumor suppressor gene. Genetic events especially, such as mitotic recombination, chromosome loss and deletion, are frequently responsible for the loss of the functional allele of heterozygous mutant tumor suppressor genes. We generated an Aprt(+/-) mouse model that allows us to study in detail the nature of the alterations that lead to loss of the wild-type Aprt allele in somatic cells. These genetic changes are thought to be analogous to those occurring at autosomal tumour suppressor genes, where they may contribute to the development of cancer. Furthermore, this mouse model allows determination of the extent and mechanisms by which chemical carcinogens induce loss of heterozygosity and identification of the nature of the DNA adducts responsible.

The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage.

In this study the role of nucleotide excision repair (NER) in protecting mouse embryonic stem (ES) cells against the genotoxic effects of UV-photolesions was analysed. Repair of cyclobutane pyrimidine dimers (CPD) in transcribed genes could not be detected whereas the removal of (6-4) photoproducts (6-4PP) was incomplete, already reaching its maximum (30%) 4 h after irradiation. Measurements of repair replication revealed a saturation of NER activity at UV doses >5 J/m2 while at a lower dose (2.5 J/m2) the repair kinetics were similar to those in murine embryonic fibroblasts (MEFs). Cytotoxic and mutagenic effects of photolesions were determined in ES cells differing in NER activity. ERCC1-deficient ES cells were hypermutable (10-fold) compared to wild-type cells, indicating that at physiologically relevant doses ES cells efficiently remove photolesions. The effect of the NER deficiency on cytoxicity was only 2-fold. Exposure to high UV doses (10 J/m2) resulted in a rapid and massive induction of apoptosis. Possibly, to avoid the accumulation of mutated cells, ES cells rely on the induction of a strong apoptotic response with a simultaneous shutting down of NER activity.