Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

IgE antibodies distinctively recognising allergenic epitopes would be ideal reagents in immunodiagnostics to detect and quantify allergens, as well as for the development of allergy diagnostics and therapeutics. We have isolated recombinant human IgE antibodies specific for the major latex allergen, hevein, from antibody phage display libraries using a green fluorescent protein (GFP)-hevein fusion as a selection antigen. Human IgE phage display libraries were constructed by combining the IgE heavy chain genes to kappa and lambda light-chain genes which were isolated from lymphocytes of a latex allergic patient. The screening of antibody libraries resulted in the enrichment of two hevein-binding scFvs designated as 1A4 and 1C2. Both antibodies showed specific binding to the hevein that could be inhibited by both the recombinant GFP-hevein and native hevein isolated from latex examination gloves. The scFvs were prone to aggregate and, thus, for further characterisation, they were converted to Fab fragments with human IgG1 or IgE isotype. Similar hevein-binding properties of the 1A4 and 1C2 Fab fragments and human IgE serum pool, conventionally used in the detection of latex allergens, demonstrate the potential utility of these recombinant antibodies for the analysis of latex allergen.     

Detection of a novel repeated sequence useful for epidemiological typing of pathogenic Yersinia enterocolitica

Strains (n = 203) of Yersinia species were used in genotyping and PCR experiments in order to evaluate the genotyping potential of the YeO:3RS probe. This probe comprises a 12.5 kb genomic fragment of the Y. enterocolitica O:3 lipopolysaccharide O-antigen gene cluster cloned into plasmid pBR322. The genotyping potential of YeO:3RS was shown to reside in the region upstream of the O-antigen gene cluster, i.e., in the first 1.65 kb of the cloned genomic fragment that contains a repeated sequence (RS) present in multiple copies in the genome. In genotyping, the YeO:3RS probe was hybridised to DNA of Yersinia enterocolitica isolates (n = 112) from humans, animals and food, along with strains of other Yersinia species (n = 5) and Salmonella enterica strains (n = 3). The YeO:3RS probe efficiently detected and subtyped all European pathogenic Yersinia enterocolitica isolates of the serobiotypes O:3/4, O:9/2 and O:5,27/2 studied (n = 87), whereas it hybridised only weakly or not at all with the other strains. Within Yersinia enterocolitica serobiotype O:3/4 strains, YeO:3RS genotyping was as discriminatory as genotyping by pulsed-field gel electrophoresis (PFGE) of XbaI-NotI digested genomic DNA. When these two methods were combined, YeO:3RS genotyping divided both of the two predominant PFGE types into six subtypes, thus increasing the discrimination. In PCR screening of additional 86 Yersinia strains, the 1.65 kb region was detected in European pathogenic serotypes O:1 and O:2 in addition to serotypes O:3, O:5,27 and O:9, indicating that it can be exploited in detecting and typing of European pathogenic serotypes in general.     

Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance

The oxidation of low density lipoproteins (LDLs) is thought to take place in the arterial intima when the particles have become isolated from circulating water-soluble antioxidants. We hypothesized that isoflavonoid antioxidants derived from soy could be incorporated into lipoproteins and possibly could protect them against oxidation, which is regarded as atherogenic. Six healthy volunteers received 3 soy bars [containing the isoflavonoid antioxidants genistein (12 mg) and daidzein (7 mg)] daily for 2 weeks. LDLs were isolated from blood drawn at the the end of a 2-week dietary baseline period, after 2 weeks on soy, and after discontinuation of soy. Large increases in plasma isoflavonoid levels occurred during soy feeding, but only minute amounts were stably associated with lipoproteins (less than 1% of plasma isoflavonoids in the LDL fraction). The LDLs were subjected to copper-mediated oxidation in vitro. Compared with off soy values, lag phases of LDL oxidation curves were prolonged by a mean of 20 min (P < 0.02) during soy intake, indicating a reduced susceptibility to oxidation. The results suggest that intake of soy-derived antioxidants, such as genistein and daidzein, may provide protection against oxidative modification of LDL. As only very small amounts of these substances were detected in purified LDL, modified LDL particles may have been produced in vivo by circulating isoflavonoids promoting resistance to oxidation ex vivo.     

High frequency of BRAF V600E mutations in ameloblastoma

Ameloblastoma is a benign but locally infiltrative odontogenic neoplasm. Although ameloblastomas rarely metastasise, recurrences together with radical surgery often result in facial deformity and significant morbidity. Development of non‐invasive therapies has been precluded by a lack of understanding of the molecular background of ameloblastoma pathogenesis. When addressing the role of ERBB receptors as potential new targets for ameloblastoma, we discovered significant EGFR over‐expression in clinical samples using real‐time RT–PCR, but observed variable sensitivity of novel primary ameloblastoma cells to EGFR‐targeted drugs in vitro. In the quest for mutations downstream of EGFR that could explain this apparent discrepancy, Sanger sequencing revealed an oncogenic BRAF V600E mutation in the cell line resistant to EGFR inhibition. Further analysis of the clinical samples by Sanger sequencing and BRAF V600E‐specific immunohistochemistry demonstrated a high frequency of BRAF V600E mutations (15 of 24 samples, 63%). These data provide novel insight into the poorly understood molecular pathogenesis of ameloblastoma and offer a rationale to test drugs targeting EGFR or mutant BRAF as novel therapies for ameloblastoma. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk     

Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer

Inherited predisposition to breast cancer is known to be caused by loss-of-function mutations in BRCA1, BRCA2, PALB2, CHEK2, and other genes involved in DNA repair. However, most families severely affected by breast cancer do not harbor mutations in any of these genes. In Finland, founder mutations have been observed in each of these genes, suggesting that the Finnish population may be an excellent resource for the identification of other such genes. To this end, we carried out exome sequencing of constitutional genomic DNA from 24 breast cancer patients from 11 Finnish breast cancer families. From all rare damaging variants, 22 variants in 21 DNA repair genes were genotyped in 3,166 breast cancer patients, 569 ovarian cancer patients, and 2,090 controls, all from the Helsinki or Tampere regions of Finland. In Fanconi anemia complementation gene M (FANCM), nonsense mutation c.5101C>T (p.Q1701X) was significantly more frequent among breast cancer patients than among controls [odds ratio (OR) = 1.86, 95% CI = 1.26–2.75; P = 0.0018], with particular enrichment among patients with triple-negative breast cancer (TNBC; OR = 3.56, 95% CI = 1.81–6.98, P = 0.0002). In the Helsinki and Tampere regions, respectively, carrier frequencies of FANCM p.Q1701X were 2.9% and 4.0% of breast cancer patients, 5.6% and 6.6% of TNBC patients, 2.2% of ovarian cancer patients (from Helsinki), and 1.4% and 2.5% of controls. These findings identify FANCM as a breast cancer susceptibility gene, mutations in which confer a particularly strong predisposition for TNBC.Breast cancer is the most common cancer affecting women worldwide. It is also the principal cause of death from cancer among women globally, accounting for 14% of all cancer deaths (1). The etiology of breast cancer is multifactorial, and the risk depends on various factors like age, family history, and reproductive, hormonal, or dietary factors. The majority of breast cancers are sporadic, but approximately 15% of cases show familial aggregation (2, 3). Since the identification of the first breast and ovarian cancer susceptibility genes breast cancer 1 and 2 (BRCA1 and BRCA2, respectively) by linkage analysis and positional cloning, several breast cancer susceptibility genes and alleles with different levels of risk and prevalence in the population have been recognized. BRCA1 and BRCA2 mutation carriers have more than 10-fold increased risk of breast cancer compared with women in the general population, and mutations in TP53, PTEN, STK11, and CDH1 have also been associated with a high lifetime risk of breast cancer in the context of rare inherited cancer syndromes (4). In addition, rare variants in genes such as checkpoint kinase 2 (CHEK2), ataxia telangiectasia mutated (ATM), and BRCA1 interacting helicase BRIP1, that confer a two- to fourfold increased risk, and in partner and localizer of BRCA2 (PALB2), with even higher risk estimates, have been found with candidate gene approaches (5, 6), and an increasing number of common low-risk loci with modest odds ratios (ORs; as much as 1.26-fold increased risk for heterozygous carriers) have been identified by genome-wide association studies (7).However, the major portion of hereditary breast cancer still remains unexplained, and many susceptibility loci are yet to be found. Exome sequencing combined with genotyping of the identified variants in case-control analysis is an effective method to recognize novel risk alleles, based on the assumption that disease-causing variants are rare and often accumulate in the protein-coding areas of the genome (8–10).Since the discovery that proteins encoded by the BRCA1 and BRCA2 breast/ovarian cancer susceptibility genes are directly involved in homologous recombination repair of DNA double-strand breaks, it has been evident that other genes involved in DNA repair are attractive breast cancer susceptibility candidates (4). Biallelic mutations in ATM gene cause rare ataxia telangiectasia disease and are associated with an increased risk for breast cancer as a result of improper DNA damage response (11). Fanconi anemia (FA) is a rare genetic disorder caused by biallelic mutations in FA genes that also participate in DNA repair. At least 15 FA genes have been identified (12). Patients with heterozygous mutations in certain FA genes have an elevated risk for various cancers, and monoallelic mutations in at least four of these genes [BRCA2, BRIP1, PALB2, and RAD51 paralog C (RAD51C)] are associated with an increased risk of breast or ovarian cancer (12, 13). Recurrent founder mutations in several cancer susceptibility genes, including the BRCA2, PALB2, and RAD51C FA genes, have been identified in the Finnish population (14–16). The PALB2 and RAD51C founder mutations have been detected at 2% frequency in Finnish breast or ovarian cancer families (15–17), whereas, in other populations, mutations in these genes are rare and often unique for each family. Founder effects in the isolated populations such as Finland or Iceland may enrich certain mutations and thus explain a significant proportion of all mutations in certain genes (18, 19). This provides an advantage in the search for novel susceptibility genes and alleles.In this study, we used exome sequencing to uncover previously unidentified recurrent breast or ovarian cancer predisposing variants in the Finnish population with a focus on DNA repair genes. Selected variants were further genotyped in a large case-control sample set. Our investigation revealed an association of a nonsense mutation (rs147021911) in an FA complementation gene, FANCM, with breast cancer, especially with triple-negative (TN) breast cancer (TNBC).     

Long-term efficacy and urological toxicity of low-dose-rate brachytherapy (LDR-BT) as monotherapy in localized prostate cancer

PurposeThe purpose of this study was to evaluate the incidence of late severe (≥Grade 3) urinary toxicity and the long-term efficacy after low-dose-rate brachytherapy (LDR-BT) in patients with localized prostate cancer (PCa).Methods and MaterialsDuring the years 1999–2008, 241 patients with PCa who underwent LDR-BT with I¹²⁵ and were followed up in Kuopio University Hospital were included to this analysis. The incidence of late severe (Grade 3) urinary toxicity and the long-term efficacy results were analyzed.ResultsAll D'Amico risk groups were represented, as 58.9%, 35.3%, and 5.8% of the patients were classified as low-, intermediate-, and high-risk patients, respectively. With a median followup of 11.4 years after implantation, the incidence of severe urinary toxicity increased throughout the followup period. The risk of Grade 3 urinary toxicity was highest among patients with higher Gleason scores (p = 0.016) and higher initial urine residual volumes (p = 0.017) and the cumulative incidence of severe urinary toxicity was 10.0%. The crude rate for transurethral prostatic resection was 5.8%. The relapse-free survival, the cause-specific survival, and the overall survival were 79.3%, 95.0%, and 66.4%, respectively.ConclusionsThe treatment was well tolerated as 90% of patients avoided any Grade 3 urinary toxicity. LDR-BT for localized PCa achieved high and durable efficacy. These results support the role of LDR-BT monotherapy as one of the valid primary treatment options for low-risk and favorable intermediate-risk patients.