Improved alkaline comet assay protocol for adherent HaCaT keratinocytes to study UVA-induced DNA damage

The comet assay is one of the well-accepted tests to measure radiation-induced DNA damage. The most commonly used protocols require single-cell suspensions that are embedded in agarose in order to perform electrophoresis. For adherently growing cells such as human HaCaT skin keratinocytes this method bears several problems. We show that trypsinization required for maintaining single-cell suspensions is prolonged after UV radiation and thereby reduces cell viability and allows partial repair, with the consequence of reduced damage detection after irradiation. Therefore, we here introduce a modified version of the comet assay where HaCaT cells are seeded onto comet slides 24 h before the assay and overlaid with agarose immediately after irradiation. Using this modification we are now able to reproducibly measure high DNA-damage levels (13-fold increase compared with controls) following irradiation with 60 J/cm² UVA as well as a dose-dependent increase of DNA damage after 10, 20 and 60 J/cm² UVA. Thus, by maintaining the cells in their natural configuration, i.e. adherently growing, we exclude several artefacts that are likely to influence the damage responses. These include: (i) trypsinization-dependent changes in cell morphology and polarity (clear lateral, i.e. adherent, and apical side of keratinocytes) which are likely of consequence for the gene-expression pattern, (ii) trypsin- and dislodgement-induced damage reducing cell viability, and (iii) the time delay between damage induction and damage evaluation to unpredictable results due to partial repair. Since these advantages pertain to all adherently growing cells, this improved protocol is not restricted to HaCaT cells but offers great potential also with all non-haematopoietic cells for obtaining accurate results and for studying repair processes in a highly reproducible manner.     

The scaling of dose with host body mass and the determinants of success in experimental cercarial infections

Experimental studies of parasite transmission can help to elucidate life cycles, measure the success of infective stages under different conditions, or test the efficacy of vaccination or other forms of protection against parasitic infection. By combining the results of experiments on a particular parasite taxon, one may also answer questions such as how experimental infection doses are chosen, or what determines infection success. Here, focusing on trematodes, analyses are conducted on data compiled from a total of 145 cercarial infection experiments (62 on non-schistosomes, 83 on schistosomes) obtained from 115 studies. All of these involved experimental exposure of individual hosts to a single known dose of cercariae under controlled laboratory conditions. Across these studies, the cercarial dose used showed a strong positive relationship with the body mass of the target host, independently of the taxonomic identity of that host or of the method of infection used. Although justification for the chosen dose was rarely given, the larger the target host, the more cercariae it was exposed to. Across all experiments, there was also evidence for a weak but significant dose-dependent effect on infection success: the higher the dose used in an experiment, the smaller the proportion of cercariae recovered from the host. This effect was mitigated by either host body mass (for schistosomes) or host taxonomic identity (for non-schistosomes), with infection being lower in fish than in other host types. Experimental procedures also impacted significantly on infection success, namely the infection method used (for schistosomes) and the time between infection and recovery of parasites (for non-schistosomes). Overall, this analysis of published experimental results provides evidence of both biological processes and confounding methodological effects, and it provides strong arguments for greater rationale in the design of experimental infection studies.     

Mathematical model of a three-stage innate immune response to a pneumococcal lung infection

Pneumococcal pneumonia is a leading cause of death and a major source of human morbidity. The initial immune response plays a central role in determining the course and outcome of pneumococcal disease. We combine bacterial titer measurements from mice infected with Streptococcus pneumoniae with mathematical modeling to investigate the coordination of immune responses and the effects of initial inoculum on outcome. To evaluate the contributions of individual components, we systematically build a mathematical model from three subsystems that describe the succession of defensive cells in the lung: resident alveolar macrophages, neutrophils and monocyte-derived macrophages. The alveolar macrophage response, which can be modeled by a single differential equation, can by itself rapidly clear small initial numbers of pneumococci. Extending the model to include the neutrophil response required additional equations for recruitment cytokines and host cell status and damage. With these dynamics, two outcomes can be predicted: bacterial clearance or sustained bacterial growth. Finally, a model including monocyte-derived macrophage recruitment by neutrophils suggests that sustained bacterial growth is possible even in their presence. Our model quantifies the contributions of cytotoxicity and immune-mediated damage in pneumococcal pathogenesis.