A new molecular subtyping approach was developed which is based on the amplification and sequencing of a repetitive region of the P1 gene of Mycoplasma pneumoniae. It allows the differentiation of all known subtypes and variants of M. pneumoniae as well as the identification of new subtypes directly in clinical samples to characterize endemic and epidemic M. pneumoniae infections.Mycoplasma pneumoniae is a common agent of infections of the human respiratory tract, with clinical courses ranging from mild forms of pharyngitis and tracheobronchitis to severe cases of interstitial pneumonia (11). Epidemiological studies have shown that M. pneumoniae is responsible for up to 50% of community-acquired pneumonias (7,15,26). Epidemic outbreaks were registered in time intervals between 3 and 7 years (16,22), leading to the hypothesis that different subtypes of M. pneumoniae with variable antigenic properties are circulating in the human population (6, 10). Although from a genetic point of view M. pneumoniae can be considered a highly uniform species, isolates could be assigned to two subtypes differing significantly in the sequences of only four adherence-related proteins (18,21,23). Among these, the 170-kDa protein P1 was identified as a major adhesin and an immunodominant protein of M. pneumoniae (9,19). The P1 gene (MPN141) (2) contains copies of repetitive elements (repMp4 and repMp2/3) (20) with subtype-specific sequence differences (Fig. 1). Recently, two variants of M. pneumoniae were described which showed sequence variations in only the repMp2/3 element of the P1 gene (4, 12). Epidemiological information regarding the subtypes of M. pneumoniae infections is based on the molecular typing of limited numbers of isolated strains (1,3,5,13,22,25). More detailed knowledge is hampered by the fact that culture of M. pneumoniae is time-consuming (up to 21 days) and is not a widely used method in routine bacteriological practice compared to serology, direct antigen tests, or PCR (17,24,26).Here we present a method for the characterization of subtypes and variants of M. pneumoniae in DNA samples from DNAs in all samples were isolated with a QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's recommendations. The subtyping protocol is based on a nested PCR to amplify part of the repMp2/3 element of the P1 gene of M. pneumoniae, followed by sequencing. The primer pairs (ThermoHybaid,
In the active orientation the DnaA protein/dnaA box complex blocks transcribing RNA polymerase. The extent of transcription termination at different dnaA boxes was used to determine in vivo their various affinities to the DnaA protein. The rate of transcription distal to the respective dnaA box was monitored by the expression of the reporter gene galK. The dnaA boxes (5'-TTATACACA and 5'-TTATCCAAA), present in oriC, showed the strongest binding affinity. The dnaA box 5'-TTTTCCACA was mutated at eight positions such that the boxes differed from the consensus sequence, 5'-TT(A/T)T(A/C)CA(A/C)A, defined so far. Based on the different properties of the dnaA boxes, a new consensus sequence was derived: 5'-(T/C)(T/C)(A/T/C)T(A/C)C(A/G)(A/C/T(A/C).
BackgroundAround 90% of melanomas are caused by exposure to ultraviolet (UV) radiation and are therefore eminently preventable. Tanning behavior is mostly initiated in early adolescence, often with the belief that it increases attractiveness; the problems related to malignant melanoma and other skin cancers are too far in the future to fathom. Given the substantial amount of time children and adolescents spend in schools, as well as with their mobile phones, addressing melanoma prevention via both of these ways is crucial. However, no school-based intervention using mobile apps has been evaluated to date. We recently released a photoaging mobile app, in which a selfie is altered to predict future appearance dependent on UV protection behavior and skin type.ObjectiveIn this pilot study, we aimed to use mobile phone technology to improve school-based melanoma prevention and measure its preliminary success in different subgroups of students with regard to their UV protection behavior, Fitzpatrick skin type and age.MethodsWe implemented a free photoaging mobile phone app (Sunface) in 2 German secondary schools via a method called mirroring. We “mirrored” the students’ altered 3-dimensional (3D) selfies reacting to touch on mobile phones or tablets via a projector in front of their whole grade. Using an anonymous questionnaire capturing sociodemographic data as well as risk factors for melanoma we then measured their perceptions of the intervention on a 5-point Likert scale among 205 students of both sexes aged 13-19 years (median 15 years).ResultsWe measured more than 60% agreement in both items that measured motivation to reduce UV exposure and only 12.5% disagreement: 126 (63.0%) agreed or strongly agreed that their 3D selfie motivated them to avoid using a tanning bed, and 124 (61.7%) to increase use of sun protection. However, only 25 (12.5%) disagreed with both items. The perceived effect on motivation was increased in participants with Fitzpatrick skin types 1-2 in both tanning bed avoidance (n=74, 71.8% agreement in skin types 1-2 vs n=50, 53.8% agreement in skin types 3-6) and increased use of sun protection (n=70, 68.0% agreement in skin types 1-2 vs n=52, 55.3% agreement in skin types 3-6), and also positively correlated with higher age.ConclusionsWe present a novel way of integrating photoaging in school-based melanoma prevention that affects the students’ peer group, considers the predictors of UV exposure in accordance with the theory of planned behavior, and is particularly effective in changing behavioral predictors in fair-skinned adolescents (Fitzpatrick skin types 1-2). Further research is required to evaluate the intervention’s prospective effects on adolescents of various cultural backgrounds.
Peptide specificity of cultured tumor-infiltrating lymphocytes (TIL) was systematically investigated in a group of HLA-A2.1+ metastatic melanoma patients consecutively referred to our department for surgical treatment. Seven samples from 6 patients were studied. All surgical specimens showed evidence of gp 100, MART-1/Melan-A and Tyrosinase gene expression as detectable by reverse PCR (rPCR). Cultured TIL from 2 patients displayed cytotoxic activity against autologous or HLA-matched EBV-transformed cells previously pulsed with MART-1/Melan-A27-35 peptide. In contrast, no CTL activity against gp100(280-288) or tyrosinase1-9 peptides could be observed. TIL were then repeatedly stimulated in vitro with the same peptides. After 6 restimulation courses at weekly intervals, specific recognition of gp100(280-288) and MART-1/Melan-A peptides was detectable in 3 and 5 TIL populations, respectively. In one case Tyrosinase1-9-specific CTL could be demonstrated. Two TIL populations from metastases resected from a melanoma patient at 6 months' distance showed a different peptide specificity pattern, and no specific CTL could be generated from simultaneously sampled peripheral blood mononuclear cells (PBMC). All peptide-specific CTL populations also displayed significant cytotoxic activity against HLA-A2.1 matched melanoma cell lines expressing the antigens under investigation. Our data indicate that CTL specific for MART-Melan-A27-35, gp100(280-288) or Tyrosinase1-9 peptides could be expanded with varying frequency from TIL derived from 4 out of 6 HLA-A2.1+ patients whose tumors expressed the genes encoding these tumor-associated antigens (TAA).
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