A novel schistosome species hosted by Planorbella (Helisoma) trivolvis is the most widespread swimmer's itch-causing parasite in Michigan inland lakes.

Cercarial dermatitis ('swimmer's itch'; SI), characterized by small itchy bumps caused by schistosome parasites of birds and mammals, is a common problem in Michigan. Research on avian schistosomes began nearly 100 years ago in Michigan inland lakes, yet scientists are still uncovering basic biological information including the identification of local snail and parasite species that cause SI. Previous research primarily focused on lakes in the northern half of Michigan's lower peninsula, although SI occurs throughout the state. We surveyed snails and snail-borne trematodes in lakes across Michigan's lower peninsula and used quantitative polymerase chain reaction analysis of filtered water samples to identify parasites to the species level, including a recently discovered parasite species that uses the snail Planorbella (Helisoma) trivolvis as its intermediate host. Most SI mitigation efforts have focused on a parasite species hosted by the snail Lymnaea catescopium ( = Stagnicola emarginata); however, lymnaeid snails and their associated schistosome species were largely restricted to northern lakes. In contrast, P. trivolvis and its associated parasite species were common in both northern and southern Michigan lakes. A third schistosome species associated with physid snails was also present at low levels in both northern and southern lakes. These results indicate that the recently discovered parasite species and its planorbid snail intermediate host may be more important drivers of Michigan SI than previously thought, possibly due to increased definitive host abundance in recent decades. These results have potentially important implications for SI mitigation and control efforts.

Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage.

Adipose-derived stem cells (ASCs) have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342) represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 µg/mL and 5 µg/mL), with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 µg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 µg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique.

The differentiation of hMSCs counteracts their migration capability and pro-angiogenic effects in vitro.

Human mesenchymal stem cells (hMSCs) have been applied therapeutically in numerous clinical trials. The pro-angiogenic effects of hMSCs, as well as their strong tumor tropism, have been shown both in vitro and in vivo. Some studies suggest using hMSCs as potential drug-carriers for tumor therapy. In previous investigations by our group, the pro-tumorigenic effects of hMSCs on head and neck squamous cell carcinoma (HNSCC) were shown. However, the influence of hMSCs on tumor vascularization as well as the possibility of its inhibition are yet to be elucidated. The cytokine patterns of the HNSCC cell line FaDu, native hMSCs (hMSCs-nat), hMSCs differentiated into adipocytes (hMSCs-adip) and osteocytes (hMSCs-ost) were evaluated. Human umbilical vein endothelial cells (HUVECs) were co-cultured with FaDu cells, hMSCs-nat, hMSCs-adip and hMSCs-ost. The capillary tube formation assay was applied. Furthermore, the migration capability of hMSCs-nat, hMSCs-adip and hMSCs-ost towards FaDu cells was measured in a Transwell system. Spheroids were cultured from hMSCs-nat, FaDu cells and DiI-labeled HUVECs. FaDu cells, hMSCs-nat, hMSCs-adip and hMSCs-ost released a wide range of cytokines and growth factors, e.g., IL-6, IL-8, IL-10, GRO and MCP. In the capillary tube formation assay, HUVECs generated significantly longer tubes after co-cultivation with hMSCs-nat as compared to HUVECs alone and FaDu. Differentiation into adipocytes and osteocytes counteracted the tube formation. The adipogenic differentiation did not alter hMSC motility, whereas osteogenic differentiation significantly inhibited hMSC migration. Generation of multi-cellular spheroids from hMSCs-nat, FaDu cells and DiI-labeled HUVECs was possible. Florescence microscopy revealed that all HUVECs were present in the spheroid core. Taken together, hMSCs-nat have a pro-angiogenic effect. The effects are counteracted by the differentiation of hMSCs towards osteogenic and adipogenic lineages. The differentiation of stem cells into different lineages may be a promising solution to generating carriers for cancer therapy without pro-tumorigenic properties.

Human mesenchymal stem cells promote cancer motility and cytokine secretion in vitro.

Interactions of human mesenchymal stem cells (hMSC) with tumors are controversially discussed since there is evidence for both tumor progression as well as tumor inhibition by hMSC. The objective of the present study is to investigate whether hMSC support cell motility and cytokine secretion in a head and neck squamous cell carcinoma cell line (HLaC 78). A spheroid model was generated in which the ultrastructure of spheroids was analyzed using scanning electron microscopy (SEM). The migration capability was monitored in a monolayer as well as in a spheroid model. The variation in migration and secretion of interleukin (IL)-6, IL-8 and vascular endothelial growth factor (VEGF), as well as the expression of the multidrug resistance gene (MDR-1) was investigated. Finally, the alteration in the cell cycle was analyzed by flow cytometry. SEM showed a tight cell-cell contact with extensive secretion of extracellular matrix. The migration and invasion capability of HLaC 78 was enhanced by hMSC. Cancer cell motility was also increased by hMSC as well as secretion of the cytokines IL-6, IL-8 and VEGF. hMSC did not induce the expression of MDR-1 in HLaC 78, and there was no alteration in the cell cycle of HLaC 78 after cocultivation with hMSC. Our results confirm the important role of hMSC in cancer biology since both an enhancement of cell motility as well as cytokine secretion could be shown. However, based on these findings and those in the current literature, caution must be applied when using hMSC as a carrier for tumor therapy in cancer treatment.

DiI labeling of human adipose-derived stem cells: evaluation of DNA damage, toxicity and functional impairment.

INTRODUCTION: Adipose tissue-derived stem cells (ASCs) have become the primary focus of tissue engineering research. To understand their functions and behavior in in vitro and in vivo models, it is mandatory to track the implanted cells and distinguish them from the resident or host cells. A common labeling method is the use of fluorescent dyes, e.g. the lipophilic carbocyanine dye, DiI. This study aimed to analyze potential DNA damage, toxicity and impairment of the functional properties of human ASCs after labeling with DiI. METHODS: Cytotoxicity was measured using the MTT assay and DNA damage was determined by means of the comet assay. Potential apoptotic effects were determined using the annexin V-propidium iodide test. Differentiation potential was evaluated by trilineage differentiation procedures in labeled and unlabeled ASCs. Proliferation as well as migration capability was analyzed, and the duration and stability of DiI labeling in ASCs during in vitro expansion was observed over a period of 35 days. RESULTS: DiI labeling did not cause genotoxic effects 15, or 30 min or 24 h after the labeling procedure, and there were no cytotoxic effects until 72 h afterwards. No impairment of proliferation or migration capability or differentiation potential could be determined. However, after 35 days, only 37% of labeled cells could be detected using the fluorescence microscope, which indicates a decrease in staining stability during in vitro expansion. CONCLUSION: DiI is a convenient method for ASCs labeling which causes no toxic effects and does not impair the proliferation, migration or differentiation potential of ASCs after the labeling procedure.

Effects of salinomycin on human bone marrow-derived mesenchymal stem cells in vitro.

Various hypotheses on the origin of cancer stem cells (CSCs) exist, including that CSCs develop from transformed human bone marrow mesenchymal stem cells (hBMSC). Since the polyether antibiotic salinomycin selectively kills CSCs, the present study aims to elucidate the effects of salinomycin on normal hBMSC. The immunophenotype of hBMSC after salinomycin exposure was observed by flow cytometry. The multi-differentiation capacity of hBMSC was evaluated by Oil Red O and van Kossa staining. Cytotoxic effects of salinomycin were monitored by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. Furthermore, spheroid formation and migration capacity were assessed. There were no differences in the immunophenotype and multi-differentiation capacity of hBMSC induced by salinomycin treatment. Cytotoxic effects were observed at concentrations of 30 µM and above. Neither the migration capability nor the ability to form spheroids was affected. Essential functional properties of hBMSC were unaffected by salinomycin. However, dose-dependent cytotoxicity effects could be observed. Overall, low dose salinomycin showed no negative effects on hBMSC. Since mesenchymal stem cells from various sources respond differently, further in vitro studies are needed to clarify the effect of salinomycin on tissue-specific stem cells.

Tracing the genetic roots of the indigenous White Park Cattle.

The White Park Cattle (WPC) is an indigenous ancient breed from the British Isles which has a long-standing history in heroic sagas and documents. The WPC has retained many primitive traits, especially in their grazing behaviour and preferences. Altogether, the aura of this breed has led to much speculation surrounding its origin. In this study, we sequenced the mitogenomes from 27 WPC and three intronic fragments of genes from the Y chromosome of three bulls. We observed six novel mitogenomic lineages that have not been found in any other cattle breed so far. We found no evidence that the WPC is a descendant of a particular North or West European branch of aurochs. The WPC mitogenomes are grouped in the T3 cluster together with most other domestic breeds. Nevertheless, both molecular markers support the primitive position of the WPC within the taurine breeds.

Ex vivo toxicity of nitrogen dioxide in human nasal epithelium at the WHO defined 1-h limit value.

Current pollution limits indicating potential harm to human health caused by nitrogen dioxide have prompted a variety of studies on the cytotoxicity and genotoxicity of nitrogen dioxide (NO2) in vitro. The present study focuses on toxic effects of NO2 at the WHO defined 1-h limit value of 200 µg NO2/m(3) air, equivalent to 0.1 ppm NO2. Nasal epithelial mucosa cells of 10 patients were cultured as an air-liquid interface and exposed to 0.1 ppm NO2 for 0.5 h, 1 h, 2 h and 3 h and synthetic air as negative control. After exposure, analysis of genotoxicity was performed by the alkaline single cell microgel electrophoresis (comet) assay and by the micronucleus test. Depression of proliferation and cytotoxic effects were checked by the micronucleus assay and the trypan blue exclusion assay. The experiments demonstrated significant DNA fragmentation even at the shortest exposure duration of half an hour in the comet assay. The amount of DNA fragmentation significantly increased with extended NO2 exposure durations. The amount of DNA fragmentation increased with extended exposure durations to synthetic air at a significantly lower level as compared to NO2 exposure. Micronucleus inductions were seen only at the longest exposure duration of 3h. There were no changes in proliferation seen in the micronucleus assay under any experimental setup. Moreover, no signs of necrosis, apoptosis or changes in viability were detected. Data demonstrate genotoxicity of NO2 at concentrations found in the urban atmosphere during short exposure durations. DNA alterations in the micronucleus assay at an exposure time of 3h indicate a significant DNA alteration possibly being hazardous to humans.

Bisphosphonate-induced osteonecrosis of the external ear canal: a retrospective study.

In 2003, osteonecrosis of the jaw was described as an intraoral complication of bisphosphonate therapy. More recently, cases of avascular necrosis of the hip were reported in patients with long-lasting bisphosphonate therapy. Thus, it was the aim of the present study to analyze cases of benign osteonecrosis of the external ear canal and to retrospectively identify a possible relationship to long-lasting bisphosphonate therapy. 13 patients with osteonecrosis of the external ear canal operated on between 2005 and 2009 were included. Patient histories were reviewed for possible previous or current bisphosphonate therapy. Three patients with osteonecrosis of the external ear canal and long-term bisphosphonate therapy could be identified. They had been treated either for breast cancer or multiple myeloma. Certainly, the jaw is an area of increased risk for developing osteonecrosis with its high mechanical stress and intraoral bacterial flora. However, osteonecrosis of the hips and the external ear canal in patients receiving long-term bisphosphonate therapy necessitate further investigation of a possible systemic, bisphosphonate-related phenomenon.

Optimization of fibrinogen isolation for manufacturing autologous fibrin glue for use as scaffold in tissue engineering.

Numerous manufacturing techniques for autogenous fibrin glue used as scaffold material have been described. As there is no consensus regarding the influence of chemical additives on cell biology, it was the aim of this study to establish a method for manufacturing autologous fibrin glue without any additives. The serum part was separated from whole blood. After fibrinogen precipitation, centrifugation was performed to obtain the fibrinogen pellet. Various experimental series were run to examine influences of various temperatures or substituting centrifugation for sedimentation. The method as described here is effective, simple, and performed without any additives, which could potentially influence cell biology.

Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium.

Cytotoxicity and genotoxicity of nitrogen dioxide (NO(2)) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO(2) in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract. Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO(2), 0.1 ppm NO(2), 1 ppm NO(2), 10 ppm NO(2) and synthetic air for half an hour. After exposure, genotoxicity was evaluated by the alkaline single-cell microgel electrophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively. The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO(2) in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO(2) in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.

Optimization of platelet isolation and extraction of autogenous TGF-beta in cartilage tissue engineering.

Platelets are enriched with Transforming Growth Factor-beta (TGF-beta). However, information is limited concerning TGF-beta's effects at the molecular level. Nevertheless, it has been demonstrated that TGF-beta activates cell proliferation and its positive influence on cartilage formation has been proven within the field of Tissue Engineering (TE). As Platelet Rich Plasma (PRP) contains TGF-beta, it was the purpose of this study to optimize PRP-isolation for further TGF-beta extraction. Red blood cell count (RBC) was separated from whole blood by centrifugation. From the supernatant PRP and platelet poor plasma (PPP) layer, the latter supernatant was re-centrifuged to extract PRP. Various experimental series were run to investigate influences concerning anticoagulating alternatives, different amounts of buffer, various centrifugal forces, or substituting centrifugation for sedimentation. TGF-beta levels were determined using ELISA. The technique of platelet-/ TGF-beta-extraction described here proves to be more effective than other methods, is easily repeatable and not time-consuming, which predisposes it for TE requirements.

Therapy of auricular keloids: review of different treatment modalities and proposal for a therapeutic algorithm.

Keloids are abnormal wound reactions of connective tissue. Auricular keloids can develop as a result of, e.g., otoplasty, ear piercing, or skin trauma. A wide variety of therapeutic options exists, including surgery as primary treatment. Furthermore, there are medical, physical, radiotherapeutic and experimental options. The present paper focuses on the different techniques including the therapeutic outcome and quality rating for each chosen pathway. In addition to the experience of the university hospitals, a thorough review of the literature was performed in order to update and compare today's therapeutic options. Surgical techniques are customized to the lesion's specific localization and extent. They may include revision of otoplasty. With medical treatment, established modalities such as steroid injection have to be distinguished from experimental methods like interferon, 5-FU, verapamil, imiquimod, or mitomycin C. Radiation is generally accepted to be effective, especially applied accompanying surgery, but needs to be restricted due to possible side effects. Physical therapy, e.g., pressure in a variety of application modalities, has gained a profound position in the therapy of auricular keloids. The success rates of the different treatment modalities vary markedly, and the number of patients per study is considerably low. Resuming the results, a periodic follow-up and good patients' compliance are mandatory to early realize and treat auricular keloids. However, studies are needed to evaluate accepted and experimental therapies including larger number of patients.

[Effects of nicotine with special consideration given to tumorigenesis in the head and neck region]. / Effekte von Nikotin unter besonderer Berücksichtigung der Tumorentstehung im Kopf-Hals-Bereich.

BACKGROUND: Tumorigenesis is based on initiation, promotion, and progression, whereas tobacco smoke is a decisive predisposing factor for squamous cell carcinomas of the upper aerodigestive tract. A variety of tobacco smoke compounds is known to potentially initiate tumors, but the alkaloid nicotine is generally considered to induce addiction only. However, there is growing evidence that nicotine may also contribute to early stages of tumorigenesis. In the present study, a possible direct genotoxic potential of nicotine is investigated. MATERIAL AND METHODS: Lymphatic tissue of the tonsilla palatina of eight donors was harvested during surgery and incubated with nicotine. DNA damage was measured with the comet assay. RESULTS: Genotoxic effects of nicotine could be demonstrated. DISCUSSION: The results suggest a direct contribution of nicotine to tumor initiation and carcinogenesis.