The Effect of Sex-Specific Differences on IL-10-/- Mouse Colitis Phenotype and Microbiota.

Sexual dimorphism is an important factor in understanding various diseases, including inflammatory bowel disease (IBD). While females typically exhibit stronger immune responses, the role of sex in IBD remains unclear. This study aimed to explore the sex-dependent differences and inflammatory susceptibility in the most extensively used IBD mouse model as they developed colitis. We monitored IL10-deficient mice (IL-10-/-) up to 17 weeks of age and characterized their colonic and fecal inflammatory phenotype, as well as their microbiota changes. Here, we originally identified IL-10-/- female mice as more prone to developing intestinal inflammation, with an increase in fecal miR-21, and dysbiosis with more detrimental characteristics compared to males. Our findings provide valuable insights into the sex-based differences in the pathophysiology of colitis and emphasize the importance of considering sex in experimental designs. Moreover, this study paves the way for future investigations aiming at addressing sex-related differences for the development of adequate disease models and therapeutic strategies, ideally enabling personalized medicine.

Fecal microbiota transplantation in a rodent model of short bowel syndrome: A therapeutic approach?

Extensive intestinal resection leads to Short Bowel Syndrome (SBS), the main cause of chronic intestinal failure. Colon preservation is crucial for spontaneous adaptation, to improve absorption and reduce parenteral nutrition dependence. Fecal microbiota transplantation (FMT), a promising approach in pathologies with dysbiosis as the one observed in SBS patients, was assessed in SBS rats with jejuno-colonic anastomosis. The evolution of weight and food intake, the lenght of intestinal villi and crypts and the composition of fecal microbiota of Sham and SBS rats, transplanted or not with high fat diet rat microbiota, were analyzed. All SBS rats lost weight, increased their food intake and exhibited jejunal and colonic hyperplasia. Microbiota composition of SBS rats, transplanted or not, was largely enriched with Lactobacillaceae, and α- and ß-diversity were significantly different from Sham. The FMT altered microbiota composition and α- and ß-diversity in Sham but not SBS rats. FMT from high fat diet rats was successfully engrafted in Sham, but failed to take hold in SBS rats, probably because of the specific luminal environment in colon of SBS subjects favoring aero-tolerant over anaerobic bacteria. Finally, the level of food intake in SBS rats was positively correlated with their Lactobacillaceae abundance. Microbiota transfer must be optimized and adapted to this specific SBS environment.

The Appendix Orchestrates T-Cell Mediated Immunosurveillance in Colitis-Associated Cancer.

BACKGROUND & AIMS: Although appendectomy may reduce colorectal inflammation in patients with ulcerative colitis (UC), this surgical procedure has been suggested to be associated with an increased risk of colitis-associated cancer (CAC). Our aim was to explore the mechanism underlying the appendectomy-associated increased risk of CAC. METHODS: Five-week-old male BALB/c mice underwent appendectomy, appendicitis induction, or sham laparotomy. They were then exposed to azoxymethane/dextran sodium sulfate (AOM/DSS) to induce CAC. Mice were killed 12 weeks later, and colons were taken for pathological analysis and immunohistochemistry (CD3 and CD8 staining). Human colonic tumors from 21 patients with UC who underwent surgical resection for CAC were immunophenotyped and stratified according to appendectomy status. RESULTS: Whereas appendectomy significantly reduced colitis severity and increased CAC number, appendicitis induction without appendectomy led to opposite results. Intratumor CD3+ and CD8+ T-cell densities were lower after appendectomy and higher after appendicitis induction compared with the sham laparotomy group. Blocking lymphocyte trafficking to the colon with the anti-α4ß7 integrin antibody or a sphingosine-1-phosphate receptor agonist suppressed the inducing effect of the appendectomy on tumors' number and on CD3+/CD8+ intratumoral density. CD8+ or CD3+ T cells isolated from inflammatory neo-appendix and intravenously injected into AOM/DSS-treated recipient mice increased CD3+/CD8+ T-cell tumor infiltration and decreased tumor number. In UC patients with a history of appendectomy, intratumor CD3+ and CD8+ T-cell densities were decreased compared with UC patients without history of appendectomy. CONCLUSIONS: In UC, appendectomy could suppress a major site of T-cell priming, resulting in a less efficient CAC immunosurveillance.

Nod2 Protects the Gut From Experimental Colitis Spreading to Small Intestine.

BACKGROUND AND AIMS: Nucleotide oligomerization domain 2 [NOD2] mutations are key risk factors for Crohn's disease [CD]. NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the exact roles of NOD2 in CD and other NOD2-associated disorders remain poorly known. METHODS: We initially observed that NOD2 expression was increased in epithelial cells away from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation, and cytokines expression were examined in the small bowel of wild-type [WT], Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulphonic acid [TNBS]. RESULTS: In WT mice, Nod2 upregulation upstream to rectal injury was associated with pro-inflammatory cytokine expression but no overt histological inflammatory lesions. Conversely, in Nod2-deficient mice the inflammation spread from colitis to ileum and duodenum. CONCLUSIONS: Nod2 protects the gut from colitis spreading to small intestine.

Is Crohn's Disease the Price to Pay Today for Having Survived the Black Death?

BACKGROUND AND AIMS: Nucleotide Oligomerisation Domain 2 [NOD2] is a key gene of innate immunity which participates in the host defence against pathogens. Several loss-of-function NOD2 mutations are associated with Crohn's disease [CD]. Their high frequencies in populations of European ancestry suggest a model of balancing selection. Because NOD2 deficiency has been associated with a resistance to Yersinia pseudotuberculosis in mice, we hypothesised that NOD2 mutations have been selected during past plague outbreaks due to the closely related bacterium Yersinia pestis. METHODS: Contemporary frequencies of the main CD-associated NOD2 mutations [R702W, G908R, and 1007fs], measured in healthy people from European and Mediterranean countries, were collected from 60 studies via a PubMed search. Plague exposure was calculated from a dataset providing outbreaks from 1346 to 1860 in Europe and the Mediterranean Bassin. A plague index was built to capture the intensity of plague exposure in the studied geographical areas. RESULTS: NOD2 mutation frequencies were associated with the past exposure to plague. Statistical significance was obtained for the most frequent mutation [R702W, p = 0.03] and for the pooled three mutations [p = 0.023]. The association remained significant when putative demographic biases were considered. CONCLUSIONS: This result argues for a selection of CD-associated NOD2 mutations by plague outbreaks and further questioned the role of exposure to enteropathogenic Yersinia species in CD.

Prevalence of Yersinia Species in the Ileum of Crohn's Disease Patients and Controls.

Yersinia are common contaminants of food products, but their prevalence in the human gut is poorly documented. Yersinia have been implicated in Crohn's Disease (CD, an inflammatory bowel disease) however their role in CD is controversial. We performed highly sensitive PCR assays of specific sequences for the gyrB gene of Y. aldovae, Y. bercovieri, Y. enterocolitica, Y. intermedia, Y. mollaretii and the inv gene of Y. pseudotuberculosis. We analyzed a total of 470 ileal samples taken from 338 participants (262 CD patients and 76 controls) belonging to three independent cohorts. All patients and controls were phenotyped and genotyped for the main CD susceptibility variants: NOD2, ATG16L1, and IRGM. Yersinia were found in 7.7% of ileal samples (respectively 7.9 and 7.6% in controls and CD patients) corresponding to 10% of participants (respectively 11.8 and 9.5% in controls and CD patients). Y. enterocolitica, Y. pseudotuberculosis and Y. intermedia were the most frequently identified species. The bacteria were more frequent in resected specimens, lymph nodes and Peyer's patches. Yersinia were no more likely to be detected in CD tissues than tissues from inflammatory and non-inflammatory controls. CD patients treated with immunosuppressants were less likely to be Yersinia carriers. In conclusion, this work shows that Yersinia species are frequently found at low levels in the human ileum in health and disease. The role of Yersinia species in this ecosystem should now be explored.

Complementary Roles of Nod2 in Hematopoietic and Nonhematopoietic Cells in Preventing Gut Barrier Dysfunction Dependent on MLCK Activity.

BACKGROUND: Crohn's disease (CD) pathogenesis is multifactorial involving genetic and environmental factors. Loss of function mutations in the nucleotide oligomerization domain 2 (NOD2) gene are the main genetic risk factor for CD. Like patients with CD, Nod2 mice are characterized by an enhanced Th1 immune response and a defective mucosal barrier function evidenced by increased intestinal permeability. We previously showed that the latter is related to hematopoietic Nod2 deficiency. Our aim was to explore the mechanisms by which Nod2 expressed in the hematopoietic and in the nonhematopoietic compartments interplay to control epithelial paracellular permeability. METHODS: Depletion of CD4 T cells in Nod2 mice and treatments with inhibitors were conducted in chimeric mice transplanted with bone marrow cells from Nod2-deficient donors into Nod2-sufficient recipients or vice versa. Caco-2 cells overexpressing a NOD2 gene which did or did not include a CD-associated polymorphism were treated with inhibitors or siRNAs and cocultured with hematopoietic cells from Peyer's patches. RESULTS: In vivo and in vitro Nod2 in hematopoietic cells regulates epithelial paracellular permeability through cytokine production influencing myosin light chain kinase (MLCK) activity. Indeed, tumor necrosis factor-α and interferon-γ secretion by CD4 T cells upregulated expression and activity of epithelial MLCK leading to increased epithelial tight junction opening. When stimulated by muramyl dipeptide, Nod2 in the nonhematopoietic compartment normalized the permeability and T-cell cytokine secretion and regulated MLCK activity. This MLCK regulation is mediated by TAK1 and RICK-dependent mechanisms. CONCLUSIONS: Our study demonstrates how hematopoietic and nonhematopoietic Nod2 regulate intestinal barrier function, improving our knowledge on the mechanisms involved in CD pathogenesis.

Nod2 Deficiency Leads to a Specific and Transmissible Mucosa-associated Microbial Dysbiosis Which Is Independent of the Mucosal Barrier Defect.

BACKGROUND AND AIMS: Crohn's disease [CD] is a complex disorder characterised by an inappropriate immune response, impaired barrier function and microbial dysbiosis. Mutations in nucleotide oligomeriation domain 2 [NOD2] are CD risk factors. Increase of intestinal permeability, CD4+ T cell infiltration, and bacterial dysbiosis are also seen in Nod2-knockout [Nod2 KO] mice. However, the specificity and relationship between these Nod2-associated abnormalities remain largely unexplored. METHODS: Wild-type [WT], Nod1-knockout [Nod1 KO] and Nod2 KO mice were analysed in parallel. Microbial composition was defined by 454-pyrosequencing of bacterial 16S rRNA genes. Mucin and antimicrobial peptide expression was assessed by RT-PCR. Cell populations from Peyer's patches were determined by flow cytometry. Ussing chambers were used to measure intestinal permeability and bacterial translocation. Finally, to explore the impact of colonisation with mother's microbiota at birth, analyses were also performed in Nod2 KO and WT mice born from WT surrogate mothers after embryo transfer. RESULTS: Nod2 KO mice exhibited colonic bacterial dysbiosis different from WT and Nod1 KO mice. Altered expression of antimicrobial peptides and mucins in ileum and colon was associated with the microbial composition. Bacterial composition of Nod2 KO and WT mice obtained by embryo transfer was similar to that observed in Nod2 KO mice, arguing for a dominant effect of Nod2 KO-associated dysbiosis. In contrast, increased levels of CD4+ T cells and gut barrier defects across Peyer's patches were specific to Nod2 deficiency and independent of Microbial dysbiosis. CONCLUSIONS: Nod2 deficiency is associated with a specific dominant dysbiosis which does not drive mucosal tissue and immune alterations.

Respective Roles of Hematopoietic and Nonhematopoietic Nod2 on the Gut Microbiota and Mucosal Homeostasis.

BACKGROUND: NOD2 mutations are associated with Crohn's disease (CD). Both CD (in human) and Nod2 deficiency (in mice) are characterized by increased mucosal CD4 T-cells, an altered permeability and a microbial dysbiosis. However, the respective roles of the gut epithelial and immune compartments on the phenotype are not known. METHODS: Microbial composition, epithelial peptide secretion, intestinal permeability, and immune cell composition of Peyer patches were studied in Nod2 knock-out mice transplanted with wild-type bone marrow cells and vice versa. RESULTS: The nonhematopoietic cells control the microbiota composition and epithelial secretion of mucins and antimicrobial peptides. These parameters are correlated with recurrent associations between bacterial species and luminal products. In contrast, Nod2 in the hematopoietic compartment regulates the epithelial permeability and the gut-associated lymphoid tissue independently of the bacterial composition. CONCLUSIONS: The immune system and the gut permeability in one hand and the microbial and epithelial peptide compositions in the other hand are separate couples of interdependent parameters, both controlled by Nod2 in either the hematopoietic or nonhematopoietic lineages.

NOD2/CARD15 gene mutations in North Algerian patients with inflammatory bowel disease.

AIM: To analyse allelic frequency of NOD2 gene variants and to assess their correlation with inflammatory bowel disease (IBD) in Algeria. METHODS: We studied 132 unrelated patients diagnosed with IBD, 86 with Crohn's disease (CD) and 46 with ulcerative colitis (UC). Data was prospectively collected between January 2011 and December 2013. The demographic and clinical characteristics were recorded for all the patients. A group of 114 healthy unrelated individuals were selected as controls. All groups studied originated from different regions of North Algeria and confirmed the Algerian origin of their parents and grandparents. Informed and written consent was obtained from each of the participants. All individuals were genotyped for the three CD-associated NOD2 variants (p.Arg(702)Trp, p.Gly908Arg and p.Leu(1007)fsinsC mutations) using the polymerase chain reaction-restriction fragment length polymorphism method. Allele and genotype frequencies in patients and control subjects were compared by χ(2) test and Fisher's exact test where appropriate. Odds ratios (OR) and 95% confidence intervals (95%CI) were also estimated. Association analyses were performed to study the influence of these variants on IBD and on clinical phenotypes. RESULTS: The p.Arg(702)Trp mutation showed the highest frequency in CD patients (8%) compared to UC patients (2%) (P = 0.09, OR = 3.67, 95%CI: 0.48-4.87) and controls (5%) (P = 0.4, OR = 1.47, 95%CI: 0.65-3.31). In CD patients allelic frequencies of p.Gly908Arg and p.Leu(1007)fsinsC variants compared to HC were 3% vs 2% (P = 0.5, OR = 1.67, 95%CI: 0.44-6.34); 2% vs 1% (P = 0.4 OR = 2.69 95%CI: 0.48-14.87 respectively). In UC patients, allelic frequencies of p.Gly908Arg and p.Leu(1007)fsinsC variants compared to HC were 1% vs 2% (P = 1, OR = 1.62, 95%CI: 0.17-4.74) and 2% vs 1% (P = 0.32, OR = 0.39, 95%CI: 0.05-2.87). The total frequency of the mutated NOD2 chromosomes was higher in CD (13%), than in HC (8%) and UC (5%). In addition, NOD2 variants were linked to a particular clinical sub-phenotype in CD in this Algerian cohort. As expected, the three NOD2 variants showed a significant association with CD but did not reach statistical significance, despite the fact that the allele frequency of NOD2 variants was in the range found in most of the European populations. This might be due to the non-exposure of the NOD2 carriers to environmental factors, required for the expression of the disease. CONCLUSION: Further analyses are necessary to study genetic and environmental factors in IBD in the Algerian population, using larger patient groups.

Pseudomonas fluorescens alters the intestinal barrier function by modulating IL-1ß expression through hematopoietic NOD2 signaling.

BACKGROUND: Ileal Crohn's disease is related to NOD2 mutations and to a gut barrier dysfunction. Pseudomonas fluorescens has also been associated with ileal Crohn's disease. The aim of this study was to determine the impact of P. fluorescens on the paracellular permeability in ileum and Peyer's patches. METHODS: To explore this question, in vivo and ex vivo experiments were performed in wild-type, Nod2, Nod2, and IL-1R mice together with in vitro analyses using the Caco-2 (epithelial) and the THP-1 (monocyte) human cell lines. RESULTS: Pseudomonas fluorescens increased the paracellular permeability of the intestinal mucosa through the secretion of IL-1ß by the immune cell populations and the activation of myosin light chain kinase in the epithelial cells. Induction of the IL-1ß pathway required the expression of Nod2 in the hematopoietic compartment, and muramyl dipeptide (a Nod2 ligand) had an inhibitory effect. CONCLUSIONS: Pseudomonas fluorescens thus alters the homeostasis of the epithelial barrier function by a mechanism similar to that previously observed for Yersinia pseudotuberculosis. This work further documents a putative role of psychrotrophic bacteria in Crohn's disease.

Digestive perianastomotic ulcerations and Crohn's disease.

BACKGROUND AND AIMS: Digestive perianastomotic ulcerations (DPAU) have been occasionally reported as late complications of neonatal or childhood surgery. METHODS: We report here a series of 14 new cases. RESULTS: Cases were revealed by severe anemia, diarrhea, abdominal pain and growth failure in average 11.5 years after surgery. Ulcerations were most often multiple (n=11), located on the upper part of ileocolonic anastomoses (n=12) and difficult to treat. No granulomas were seen but lymphoid follicles were frequent. In addition, either ASCA or ANCA were positive in 4/9 tested patients and 8/11 genotyped patients exhibited a NOD2 mutation (P<0.0002 when compared to French healthy controls). CONCLUSION: Altogether, these findings argue for common physiopathological features between DPAU and Crohn's disease and for a prospective follow-up of selected operated children to explore the early events involved in gut inflammatory lesions.

Characterization of Crohn disease in X-linked inhibitor of apoptosis-deficient male patients and female symptomatic carriers.

BACKGROUND: Crohn disease is an inflammatory bowel disease (IBD) with a complex mode of inheritance. Although nucleotide binding and oligomerization domain containing 2 (NOD2) is the strongest risk factor, the cause of Crohn disease remains unknown in the majority of the cases. X-linked inhibitor of apoptosis (XIAP) deficiency causes X-linked lymphoproliferative syndrome type 2. IBD has been reported in some XIAP-deficient patients. OBJECTIVE: We characterize the IBD affecting a large cohort of patients with mutations in XIAP and examine the possible pathophysiologic mechanisms. METHODS: We performed a phenotypical and histologic analysis of the IBD affecting 17 patients with hemizygous mutations in XIAP, including 3 patients identified by screening 83 patients with pediatric-onset IBD. The X chromosome inactivation was analyzed in female carriers of heterozygous XIAP mutations, including 2 adults with IBD. The functional consequences of XIAP deficiency were analyzed. RESULTS: Clinical presentation and histology of IBD in patients with XIAP deficiency overlapped with those of patients with Crohn disease. The age at onset was variable (from 3 months to 41 years), and IBD was severe and difficult to treat. In 2 patients hematopoietic stem cell transplantation fully restored intestinal homeostasis. Monocytes of patients had impaired NOD2-mediated IL-8 and monocyte chemoattractant protein 1 (MCP-1) production, as well as IL-10, in response to NOD2 and Toll-like receptor 2/4 costimulation. Nucleotide binding and oligomerization domain containing 1 (NOD1)-mediated IL-6 and IL-8 production was defective in fibroblasts from XIAP-deficient patients. The 2 heterozygous female carriers of XIAP mutations with IBD displayed abnormal expression of the XIAP mutated allele, resulting in impaired activation of the NOD2 pathway. CONCLUSION: IBD in patients with XIAP deficiency is similar to Crohn disease and is associated with defective NOD2 function in monocytes. Importantly, we report that it is not restricted to male patients because we identified 2 symptomatic female heterozygous carriers of XIAP mutations.

Yersinia pseudotuberculosis disrupts intestinal barrier integrity through hematopoietic TLR-2 signaling.

Intestinal barrier function requires intricate cooperation between intestinal epithelial cells and immune cells. Enteropathogens are able to invade the intestinal lymphoid tissue known as Peyer's patches (PPs) and disrupt the integrity of the intestinal barrier. However, the underlying molecular mechanisms of this process are poorly understood. In mice infected with Yersinia pseudotuberculosis, we found that PP barrier dysfunction is dependent on the Yersinia virulence plasmid and the expression of TLR-2 by hematopoietic cells, but not by intestinal epithelial cells. Upon TLR-2 stimulation, Y. pseudotuberculosis-infected monocytes activated caspase-1 and produced IL-1ß. In turn, IL-1ß increased NF-κB and myosin light chain kinase activation in intestinal epithelial cells, thus disrupting the intestinal barrier by opening the tight junctions. Therefore, Y. pseudotuberculosis subverts intestinal barrier function by altering the interplay between immune and epithelial cells during infection.

Yersinia pseudotuberculosis effector YopJ subverts the Nod2/RICK/TAK1 pathway and activates caspase-1 to induce intestinal barrier dysfunction.

Yersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y. pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide. YopJ, but not the catalytically inactive mutant YopJ(C172A), acetylates critical sites in the activation loops of the RICK and TAK1 kinases, which are central mediators of Nod2 signaling, and decreases the affinity of Nod2 for RICK. Concomitantly, Nod2 interacts with and activates caspase-1, resulting in increased levels of IL-1ß. Finally, IL-1ß within PP plays an essential role in inducing intestinal barrier dysfunction. Thus, YopJ alters intestinal permeability and promotes the dissemination of Yersinia as well as commensal bacteria by exploiting the mucosal inflammatory response.

Requirement of cellular prion protein for intestinal barrier function and mislocalization in patients with inflammatory bowel disease.

BACKGROUND & AIMS: Cell adhesion is one function regulated by cellular prion protein (PrP(c)), a ubiquitous, glycosylphosphatidylinositol-anchored glycoprotein. PrP(c) is located in cell-cell junctions and interacts with desmosome proteins in the intestinal epithelium. We investigated its role in intestinal barrier function. METHODS: We analyzed permeability and structure of cell-cell junctions in intestine tissues from PrP(c) knockout (PrP(c-/-)) and wild-type mice. PrP(c) expression was knocked down in cultured human Caco-2/TC7 enterocytes using small hairpin RNAs. We analyzed colon samples from 24 patients with inflammatory bowel disease (IBD). RESULTS: Intestine tissues from PrP(c-/-) mice had greater paracellular permeability than from wild-type mice (105.9 ± 13.4 vs 59.6 ± 10.1 mg/mL fluorescein isothiocyanate-dextran flux; P < .05) and impaired intercellular junctions. PrP(c-/-) mice did not develop spontaneous disease but were more sensitive than wild-type mice to induction of colitis with dextran sulfate (32% mortality vs 4%, respectively; P = .0033). Such barrier defects were observed also in Caco-2/TC7 enterocytes following PrP(c) knockdown; the cells had increased paracellular permeability (1.5-fold over 48 hours; P < .001) and reduced transepithelial electrical resistance (281.1 ± 4.9 vs 370.6 ± 5.7 Ω.cm(2); P < .001). Monolayer shape and cell-cell junctions were altered in cultures of PrP(c) knockdown cells; levels of E-cadherin, desmoplakin, plakoglobin, claudin-4, occludin, zonula occludens 1, and tricellulin were decreased at cell contacts. Cell shape and junctions were restored on PrP(c) re-expression. Levels of PrP(c) were decreased at cell-cell junctions in colonic epithelia from patients with Crohn's disease or ulcerative colitis. CONCLUSIONS: PrP(c) regulates intestinal epithelial cell-cell junctions and barrier function. Its localization is altered in colonic epithelia from patients with IBD, supporting the concept that disrupted barrier function contributes to this disorder.

Small-molecule drugs mimicking DNA damage: a new strategy for sensitizing tumors to radiotherapy.

PURPOSE: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. EXPERIMENTAL DESIGN: A novel strategy for inhibiting DNA repair was tested. We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. RESULTS: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1 and p53-binding protein 1 repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of "false" DNA damage signaling and repair inhibition. CONCLUSIONS: These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger "false" signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.