Measurement of canine Th17 cells by flow cytometry.

Th17 cells are T helper cells which play an important role during inflammation and autoimmune disease. To investigate the role of these cells in diseases in dogs in a clinical setting, methods for fast identification had to be established. Th17 cells are a rare cell population, for their measurement stimulation is recommended. To examine more samples simultaneously and to receive a relatively high purity of cell population of CD3 + CD4+ cells, different methods on various levels of preselection of cells as well as the possibility of storing blood overnight for measuring Th17 cells by flow cytometry were investigated. Firstly, to receive a high number of mononuclear cells, two different density gradients were compared and analysed. Furthermore, the enrichment of CD3 + CD4+ cells via depletion of CD8alpha+, CD11b + and CD21+ cells by cell sorting (autoMACS Pro Separator) was tested. It was also investigated whether stimulation processes led to better interpretation of results and whether there was a significant difference in measurement of directly processed blood samples and samples that had been stored overnight. In conclusion, the use of the density gradient (Lymph24+ Spin Medium) resulted in a purer cell population through a significant decrease in polymorphonuclear cells (*p = 0.01). After cell sorting, a significant difference in cell population purity was detected. Within the target fraction (containing mainly CD3 + CD4+ cells), CD8alpha+, CD21+, CD11b + cell percentages were significantly lower (***p < 0.001, *p < 0.02, ***p < .0001, respectively), and CD3 + CD4+ cell percentage was significantly higher (***p < .0001). There was a significant difference in Th17 cell percentage between unstimulated and stimulated cell populations (***p < .0001), but no significant difference in the percentage of unstimulated Th17 cells (p = 0.29) or stimulated Th17 cells (p = 0.71) in stored blood in comparison to directly processed EDTA blood samples. Finally, a modified protocol that offers an efficient way to investigate samples that were stored overnight by means of flow cytometry was evolved to research the role of Th17 cells in dogs with different diseases or in healthy populations.

Comparative analysis of biofilm models to determine the efficacy of antimicrobials.

Biofilms are one of the greatest challenges in today's treatment of chronic wounds. While antimicrobials kill platonic bacteria within seconds, they are rarely able to harm biofilms. In order to identify effective substances for antibacterial therapy, cost-efficient, standardized and reproducible models that aim to mimic the clinical situation are required. In this study, two 3D biofilm models based on human plasma with immune cells (lhBIOM) or based on sheep blood (sbBIOM) containing S. aureus or P. aeruginosa, are compared with the human biofilm model hpBIOM regarding their microscopic structure (scanning electron microscopy; SEM) and their bacterial resistance to octenidine hydrochloride (OCT) and a sodium hypochlorite (NaOCl) wound-irrigation solution. The three analyzed biofilm models show little to no reaction to treatment with the hypochlorous solution while planktonic S. aureus and P. aeruginosa cells are reduced within minutes. After 48 h, octenidine hydrochloride manages to erode the biofilm matrix and significantly reduce the bacterial load. The determined effects are qualitatively reflected by SEM. Our results show that both ethically acceptable human and sheep blood based biofilm models can be used as a standard for in vitro testing of new antimicrobial substances. Due to their composition, both fulfill the criteria of a reality-reflecting model and therefore should be used in the approval for new antimicrobial agents.

PLS3 sequencing in childhood-onset primary osteoporosis identifies two novel disease-causing variants.

Altogether 95 children with primary bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Two children with multiple peripheral and spinal fractures and low BMD had novel disease-causing PLS3 variants. Children with milder phenotypes had no pathogenic variants. PLS3 screening is indicated in childhood-onset primary osteoporosis. INTRODUCTION: The study aimed to determine the role of pathogenic PLS3 variants in children's bone fragility and to elucidate the associated phenotypic features. METHODS: Two cohorts of children with bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Cohort I comprised 31 patients with childhood-onset primary osteoporosis of unknown etiology. Cohort II comprised 64 children who had sustained multiple fractures but were otherwise healthy. Clinical and radiological data were reviewed. Peripheral blood DNA was Sanger sequenced for coding exons and flanking intronic regions of PLS3. RESULTS: In two patients of cohort I, where other common genetic causes had been excluded, we identified two novel disease-causing PLS3 variants. Patient 1 was a male with bilateral femoral fractures at 10 years, low BMD (Z-score -4.1; 18 years), and multiple vertebral compression fractures. He had a novel nonsense variant in PLS3. Patient 2 was a girl with multiple long bone and vertebral fractures and low BMD (Z-score -6.6 at 6 years). She had a de novo missense variant in PLS3; whole exome sequencing and array-CGH identified no other genetic causes. Iliac crest bone biopsies confirmed low-turnover osteoporosis in both patients. In cohort II, no pathogenic PLS3 variants were identified in any of the subjects. CONCLUSIONS: Two novel disease-causing variants in PLS3 were identified in a boy and a girl with multiple peripheral and spinal fractures and very low BMD while no pathogenic variants were identified in children with less severe skeletal fragility. PLS3 screening is warranted in male and female patients with childhood-onset primary osteoporosis.

Anomalous muscle belly of the flexor digitorum superficialis associated with carpal tunnel syndrome: case report.

A patient had a 10-year history of pain and swelling in the right wrist and palm. Carpal tunnel exploration showed anomalous muscle bellies of flexor digitorum superficialis II and III. The muscle bellies were excised. Postoperatively, the symptoms disappeared. Our case is compared with others in the literature.

[Illnesses in relation to swimming pool visits--conclusions of a survey]. / Erkrankungen im Zusammenhang mit Schwimmbadbesuchen--Ergebnis einer Befragung.

Diseases acquired by using swimming-pools were recorded by inquiring visitors and local physicians in a German spa. The diseases were brought into relation with microbiological and chemical parameters of the swimming-pool water. Water quality was in general satisfying, but on days with a very great number of visitors the treatment-plant quickly was working beyond capacity. 663 out of 1056 visitors indicated diseases which they traced back to the visit of the swimming-pool. The quality of the water was the reason for only a part of the diseases. Eye-irritations and colds counted up to nearly half of the illnesses, other frequent complaints concerned the skin and the ear-nose-and throat-region.