IntroductionAlthough the presence of pathogens in skin wounds is known to delay the wound healing process, the mechanisms underlying this delay remain poorly understood. In the present study, we have investigated the regulatory role of proinflammatory cytokines on the healing kinetics of infected wounds.MethodsWe have developed a mouse model of cutaneous wound healing, with or without wound inoculation with Staphylococcus aureus and Pseudomonas aeruginosa, two major pathogens involved in cutaneous wound bacterial infections.ResultsAseptic excision in C57BL/6 mouse skin induced early expression of IL-1β, TNFα and Oncostatin M (OSM), without detectable expression of IL-22 and IL-17A/F. S. aureus and P. aeruginosa wound inoculation not only increased the expression of IL-1β and OSM, but also induced a strong cutaneous expression of IL-22, IL-17A and IL-17F, along with an increased number of infiltrating IL-17A and/or IL-22-producing γδ T cells. The same cytokine expression pattern was observed in infected human skin wounds. When compared to uninfected wounds, mouse skin infection delayed the wound healing process. Injection of IL-1α, TNFα, OSM, IL-22 and IL-17 together in the wound edges induced delayed wound healing similar to that induced by the bacterial infection. Wound healing experiments in infected Rag2KO mice (deficient in lymphocytes) showed a wound healing kinetic similar to uninfected Rag2KO mice or WT mice. Rag2KO infected-skin lesions expressed lower levels of IL-17 and IL-22 than WT, suggesting that the expression of these cytokines is mainly dependent on γδ T cells in this model. Wound healing was not delayed in infected IL-17R/IL-22KO, comparable to uninfected control mice. Injection of recombinant IL-22 and IL-17 in infected wound edges of Rag2KO mice re-establish the delayed kinetic of wound healing, as in infected WT mice.ConclusionThese results demonstrate the synergistic and specific effects of IL-22 and IL-17 induced by bacterial infection delay the wound healing process, regardless of the presence of bacteria per se. Therefore, these cytokines play an unexpected role in delayed skin wound healing.

Abstract.Background: BRCA1 and BRCA2 germline mutations predispose heterozygous carriers to hereditary breast/ovarian cancer. However, unclassified variants (UVs) (variants with unknown clinical significance) and missense polymorphisms in BRCA1 and BRCA2 genes pose a problem in genetic counseling, as their impact on risk of breast and ovarian cancer is still unclear. The objective of our study was to identify UVs and missense polymorphisms in Algerian breast/ovarian cancer patients and relatives tested previously for BRCA1 and BRCA2 genes germline mutations analysis. Methods: We analyzed 101 DNA samples from 79 breast/ovarian cancer families. The approach used is based on BRCA1 and BRCA2 sequence variants screening by SSCP or High-Resolution Melting (HRM) curve analysis followed by direct sequencing. In silico analyses have been performed using different bioinformatics programs to individualize genetics variations that can disrupt the BRCA1 and BRCA2 genes function. Results: Among 80 UVs and polymorphisms detected in BRCA1/2 genes (33 BRCA1 and 47 BRCA2), 31 were new UVs (10 BRCA1 and 21 BRCA2), 7 were rare UVs (4 BRCA1 and 3 BRCA2) and 42 were polymorphic variants (19 BRCA1 and 23 BRCA2). Moreover, 8 new missense UVs identified in this study: two BRCA1 (c.4066C>A/p.Gln1356Lys, c.4901G>T/p.Arg1634Met) located respectively in exons 11 and 16, and six BRCA2 (c.1099G>A/p.Asp367Asn, c.2636C>A/p.Ser879Tyr, c.3868T>A/p.Cys1290Ser, c.5428G>T/p.Val1810Phe, c.6346C>G/p.His2116Asp and c.9256G>A/p.Gly3086Arg) located respectively in exons 10, 11 and 24, show a damaging PSIC score yielded by PolyPhen2 program and could be pathogenic. In addition, 5 new BRCA2 missense UVs out of six that were found to be damaging by PolyPhen2 program, also were deleterious according to SIFT program. The rare BRCA1 UV c.5332G>A/p.Asp1778Asn was found here for the first time in co-occurrence in trans with the deleterious BRCA1 mutation c.798 799delTT/p.Ser267LysfsX19 in young breast cancer patient. Moreover, 10 new identified intronic variants with unknown clinical significance (3 BRCA1 and 7 BRCA2) in the present study, could be considered as benign, because GeneSplicer, SpliceSiteFinder and MaxEntScan prediction programs show no splice site alteration for these variants. Several missense polymorphisms of BRCA1 c.2612C>T/p.Pro871Leu, c.3548A>G/p.Lys1183Arg, c.4837A>G/p.Ser1613Gly and BRCA2 c.865A>C/p.Asn289His, c.1114A>C/p.Asn372His, c.2971A>G/p.Asn991Asp, c.7150C>A/p.Gly2384Lys have been identified with high frequency in patients who were tested negative for BRCA1 and BRCA2 mutations. These missense polymorphisms could have a role as susceptibility breast cancer markers in Algerian breast/ovarian cancer families where pathological BRCA1 and BRCA2 mutations were not present. Conclusions: For the first time, UVs and missense polymorphisms in BRCA1 and BRCA2 genes have been identified in Algerian breast/ovarian cancer families. Evaluation of breast/ovarian cancer risk induced by the eight new missense UVs and common polymorphisms detected in ou...

Background: BRCA1 and BRCA2 germline mutations predispose heterozygous carriers to hereditary breast/ovarian cancer. Unclassified variants (UVs) and missense polymorphisms in BRCA genes pose a problem in genetic counseling, as their impact on risk of breast and ovarian cancer is still unclear. The objective of our present study is to characterize UVs and missense polymorphisms in Algerian breast/ ovarian cancer patients and relatives tested previously for BRCA1/2 genes germline mutations. Methods: We analyzed 86 DNA samples from 70 breast/ovarian cancer families. The approach used is based on BRCA1/2 sequence variants screening by High-Resolution Melting (HRM) curve analysis followed by direct sequencing. To identify no synonymous amino acid changes likely to disrupt BRCA1/2 genes function, we used a comparative evolutionary bioinformatic program, Polymorphism Phenotyping 2 (PolyPhen-2). We used GeneSplicer program to identify the splice site alterations of new UVs occurring in intron-exon boundaries of BRCA1 and BRCA2 genes. Results: 76 unclassified variants and polymorphisms were detected in BRCA1/2 genes (18 BRCA1 and 58 BRCA2). 8 new missense UVs identified in the present study: two BRCA1 (c.4066C>A/p.Gln1356Lys, c.4901G>T/p.Arg1634Met) located respectively in exons 11 and 16, and six BRCA2 (c.1099G>A/p.Asp367Asn, c.2636C>A/p.Ser879Tyr, c.3868T>A/p.Cys 1290 Ser, c.5428G>T/ p.Val1810Phe, c.6346C>G/ p.His2116Asp and c.9256G>A/ p.Gly3086Arg) located respectively in exons 10, 11 and 24, show a damaging PSIC score yielded by PolyPhen-2 program and could be pathogenic. Interestingly, the new BRCA2 UV c.6346C>G/pHis2116Asp had been detected in breast/ovarian cancer patient (tested negative for a BRCA mutation) but not in her sister (diagnosed with breast cancer) who carries the BRCA1 mutation c.83_84delTG. The rare BRCA1 UV c.5332G>A/p.Asp1778Asn was found here for the first time in co-occurrence in trans with the deleterious BRCA1 mutation c.798_799delTT in young breast cancer patient. In addition, 10 new identified UVs (3 BRCA1 and 7 BRCA2) occurring in intron-exon boundaries could be considered as benign, because the GeneSplicer prediction program shows no splice alteration site for these variants. Common polymorphisms BRCA1 Gln356Arg, Pro871Leu, Glu1038Gly, Lys1183Arg, Ser1613Gly and BRCA2 Asn289His, Asn372His, p.Asn991Asp have been identified with high frequency in patients who tested negative for BRCA mutations. These missense polymorphisms could have a role as susceptibility breast cancer markers in Algerian BRCAX families. Conclusions: UVs and missense polymorphisms in BRCA1/2 genes have been characterized in Algerian breast/ovarian cancer families. Evaluation of risk of breast/ovarian cancer by the eight new missense UVs and common polymorphisms identified in our present work is on going in a larger study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5613. doi:10.1158/1538-7445.AM2011-5613

Background: Breast cancer is the leading cause of cancer death in women in Algeria. To date, few molecular genetics studies of BRCA1 and BRCA2 germline mutations have been reported in the Algerian population. The most frequent mutations encountered in BRCA genes are deletions or insertions of a few bases or single-base substitutions that result in premature stop codons. However, an increasing number of large genomic rearrangements (LGRs) have been identified especially in BRCA1 gene, whereas the frequency of genomic rearrangements in BRCA2 gene is low in many populations. The objective of our study was to characterize genomic rearrangements in 76 Algerian breast/ovarian cancer patients and relatives tested negative for small point mutations in BRCA1 and BRCA2 genes. Methods: MLPA was performed on genomic DNA as described by Schouten et al (2002). Screening for genomic rearrangements in BRCA1 and BRCA2 genes of 76 patients and relatives were performed by using MLPA commercial kits P002-B1, P087-B1 and PO45-B2, PO90-B2 respectively (MRC-Holland, Amsterdam, The Netherlands). To confirm the rearrangement and to determine the exact site of its genomic breakpoints, we performed long-range PCR of genomic DNA using the Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany). Results: MLPA analysis of the BRCA1 gene revealed two germline alterations in two unrelated patients among 76. A novel deletion of BRCA1 exon 2 (c.-19-α_80+−del/p.α) and a novel genomic breakpoint in deletion of BRCA1 exon 8 (c.442-α_547+−del/p≤) were identified in two patients with breast/ovarian cancer and bilateral breast cancer, respectively. No LGRs were detected in BRCA2 gene. We found by using Long range PCR technique that our patient with the BRCA1 exon 8 deletion is heterozygous for a 2.6 kb deletion. Investigations aimed at determining the genomic breakpoint of the BRCA1 exon 2 deletion described in our study is on going. Conclusion: For the first time, we report here two genomic rearrangements in BRCA1 gene in Algerian breast/ovarian cancer patients. Our results reinforce the idea that breast/ovarian cancer families tested negatively for small point mutations should be routinely screened with MLPA for large genomic rearrangements in BRCA1 and BRCA2 genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2595. doi:1538-7445.AM2012-2595