The ATPase activity of MLH1 is required to orchestrate DNA double-strand breaks and end processing during class switch recombination

Antibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are similarly initiated in B cells with the generation of U:G mismatches by activation-induced cytidine deaminase but differ in their subsequent mutagenic consequences. Although SHM relies on the generation of nondeleterious point mutations, CSR depends on the production of DNA double-strand breaks (DSBs) and their adequate recombination through nonhomologous end joining (NHEJ). MLH1, an ATPase member of the mismatch repair (MMR) machinery, is emerging as a likely regulator of whether a U:G mismatch progresses toward mutation or DSB formation. We conducted experiments on cancer modeled ATPase-deficient MLH1G67R knockin mice to determine the function that the ATPase domain of MLH1 mediates in SHM and CSR. Mlh1(GR/GR) mice displayed a significant decrease in CSR, mainly attributed to a reduction in the generation of DSBs and diminished accumulation of 53BP1 at the immunoglobulin switch regions. However, SHM was normal in these mice, which distinguishes MLH1 from upstream members of the MMR pathway and suggests a very specific role of its ATPase-dependent functions during CSR. In addition, we show that the residual switching events still taking place in Mlh1(GR/GR) mice display unique features, suggesting a role for the ATPase activity of MLH1 beyond the activation of the endonuclease functions of its MMR partner PMS2. A preference for switch junctions with longer microhomologies in Mlh1(GR/GR) mice suggests that through its ATPase activity, MLH1 also has an impact in DNA end processing, favoring canonical NHEJ downstream of the DSB. Collectively, our study shows that the ATPase domain of MLH1 is important to transmit the CSR signaling cascade both upstream and downstream of the generation of DSBs

Fibroblast Growth Factor Receptor 3 Mutations in Bladder Tumors Correlate with Low Frequency of Chromosome Alterations

The aim of this study was to analyze the distribution of FGFR3 mutations in bladder tumors of different grade and stage and determine the relation of mutations to chromosomal alterations detected by comparative genomic hybridization (CGH). One hundred bladder cancer samples served as templates for manual microdissection. DNA was isolated from dissected samples containing at least 80% tumor cells. Mutations in FGFR3 were analyzed by SNaPshot analysis. CGH was carried out according to standard protocols. FGFR3 mutations were detected in 45 of 92 samples (48.9%). Concerning T-category, the following mutation frequencies occurred: pTa, 69%; pT1, 38%; and pT2-3, 0%. The mutation frequency was significantly associated with tumor grade: G1, 72%; G2, 56%; and G3, 4%. In pTaG1 tumors, mutations were found in 74%. A significantly lower number of genetic alterations per tumor detected by CGH was associated with FGFR3 mutations (2 vs 8). This association was also seen in pTaG1 tumors: 2.5 (with mutation) vs 7.5 (without mutation). FGFR3 mutations characterize noninvasive low-risk tumors of low malignancy. The low malignant potential of these tumors is underlined by a low number of genetic alterations per tumor. Therefore, FGFR3 represents a valuable prognostic marker of tumors with low malignant potential and can be used as surrogate marker for the detection of genetically stable bladder tumors

Lessons learned from the implementation of an online infertility community into an IVF clinic's daily practice

The Internet is expected to innovate healthcare, in particular patient-centredness of care. Within fertility care, information provision, communication with healthcare providers and support from peers are important components of patient-centred care. An online infertility community added to an in vitro fertilisation or IVF clinic's practice provides tools to healthcare providers to meet these. This study's online infertility community facilitates peer-to-peer support, information provision to patients and patient provider communication within one clinic. Unfortunately, these interventions often fail to become part of clinical routines. The analysis of a first introduction into usual care can provide lessons for the implementation in everyday health practice. The aim was to explore experiences of professionals and patients with the implementation of an infertility community into a clinic's care practice. We performed semi-structured interviews with both professionals and patients to collect these experiences. These interviews were analyzed using the Normalisation Process Model. Assignment of a community manager, multidisciplinary division of tasks, clear instructions to staff in advance and periodical evaluations could contribute to the integration of this online community. Interviews with patients provided insights into the possible impact on daily care. This study provides lessons to healthcare providers on the implementation of an online infertility community into their practice

FGFR3 and PIK3CA mutations are involved in the molecular pathogenesis of solar lentigo

Solar lentigines (SL) are frequent benign skin lesions appearing on sun-exposed areas especially in elderly people and therefore represent a hallmark of (photo)aged skin. It has been proposed that SL may subsequently evolve into adenoid seborrhoeic keratosis (SK). However, little is known about the genetic basis of SL. In human SK, FGFR3 and PIK3CA mutations have recently been identified. To analyse SL for potential FGFR3 and PIK3CA mutations. We screened 30 SL for FGFR3 mutations using a SNaPshot((R)) multiplex assay. For PIK3CA mutations we used direct sequencing of exon 9 and a SNaPshot((R)) assay for the H1047R hotspot mutation (exon 20). Because psoralen plus ultraviolet A (PUVA) lentigines show the V600E BRAF hotspot mutation, we additionally investigated this mutation in SL by allele-specific polymerase chain reaction. FGFR3 mutations were detected in five of 30 (17%) SL and PIK3CA mutations in two of 28 (7%) SL. None of 28 SL available for BRAF analysis revealed the V600E mutation. Our results suggest that FGFR3 and PIK3CA mutations are involved in the pathogenesis of SL. The occurrence of these mutations in both SL and SK suggests a common genetic basis. Our findings furthermore substantiate previous speculations that UV exposure may be a causative factor for FGFR3 and PIK3CA mutations in human skin

Induction of CAMEL/NY-ESO-ORF2-specific CD8+ T cells upon stimulation with dendritic cells infected with a modified Ad5 vector expressing a chimeric Ad5/35 fiber

Delivery of the full-length tumor antigen might be more successful in immunotherapy than single peptides and has the advantage that patients no longer need to be selected for their HLA type. In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by dendritic cells infected with an adenovirus (Ad) type 5 vector containing the fiber shaft and knob of human serotype Ad35 (Ad5F35 vector). Our data show induction of CD8(+) T cells specific for the known HLA-A(*)0201-binding CAMEL/NY-ESO-ORF2(1-11) epitope by DC infected with Ad5F35-CAMEL, but not by DC pulsed with the recombinant CAMEL protein. In one healthy donor, even CD8(+) T cells specific for a new HLA-B7-binding CAMEL/NY-ESO-ORF2(46-54) epitope were raised. In conclusion, the in vitro induction of CAMEL/NY-ESO-ORF2-specific CD8(+) T cells in healthy donors by DC infected with Ad5F35-CAMEL strongly supports further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the generation of tumor antigen-specific CD8(+) T cell responses in viv