Microscopic evaluation of tongue dorsum biofilm from halitosis patients: an ex vivo study using confocal laser scanning microscopy (CLSM)

A category of oral biofilm which is still not well understood is the one coating the tongue, although various reports have associated its presence with halitosis in patients (1). The aim of the study was to visualize the three-dimensional bacteria distribution within the biofilm in order to better understand the ecological balance which regulates it. Tongue plaque samples from four halitosis-diagnosed patients and four healthy volunteers were analysed and compared. The biofilm was collected using a 0.1ml sterile inoculating loop. The visualization of the tongue dorsum biofilm was performed combining fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) (2). Eubacteria, Streptococcus spp. and Fusobacterium nucleatum were stained using specific fluorescent genetic probes. Morphological analysis by CLSM illustrated the different distribution of the species which were tracked: Streptococcus spp. appeared immerged within the samples, while F. nucleatum was found in the peripheral areas of the samples. Furthermore, F. nucleatum appeared to exist without the presence of the Streptococcus spp. in the halitosis group. This study showed the architecture of tongue dorsum biofilm by means of imaging techniques, highlighting the distribution of the tracked bacterial species within the biofilm sample of the plaque.The authors are grateful to Dr. A. Zurcher and to Mr. G. Heuzeroth, University of Basel, for their help in the recruiting and sampling procedures

Cost-effective fabrication of photopolymer molds with multi-level microstructures for pdms microfluidic device manufacture

This paper describes a methodology of photopolymer mold fabrication with multi-level microstructures for polydimethylsiloxane (PDMS) microfluidic device manufacture. Multi-level microstructures can be performed by varying UVA exposure time and channel width. Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and profilometry techniques have been employed to characterize the molds. Multiple molds with multi-level microstructures can be formed in a unique piece. Overall height/depth of the structures reaches up to 677 μm and a minimum of 21 μm. The method provides several advantages such as reduction of fabrication time, multiple structures with diverse topologies, a great variety of depth and height in a single mold and low cost of fabrication. The effectiveness of multi-level microstructure fabrication was evaluated by constructing PDMS microfluidic devices for cell culture and proliferation.Fil: Olmos Carreno, Carol Maritza. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Penãherrera, Ana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Albert Ludwigs University of Freiburg; AlemaniaFil: Rosero Yánez, Gustavo Ivan. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Albert Ludwigs University of Freiburg; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vizuete, Karla. Universidad de Las Fuerzas Armadas; EcuadorFil: Ruarte, Darío. Albert Ludwigs University of Freiburg; AlemaniaFil: Follo, Marie. Albert Ludwigs University of Freiburg; AlemaniaFil: Vaca Mora, Andrea Vanessa. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Arroyo, Carlos R.. Universidad de Las Fuerzas Armadas; EcuadorFil: Debut, Alexis. Universidad de Las Fuerzas Armadas; EcuadorFil: Cumbal Flores, Luis. Universidad de Las Fuerzas Armadas; EcuadorFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Florida International University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Florida International University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mertelsmann, Roland. Albert Ludwigs University of Freiburg; Alemani

Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP. YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co‐transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10−4 pmol of siRNA, and 1 × 10−3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo

The Nlrp3 inflammasome regulates acute graft-versus-host disease

The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication

Existence of reprogrammed lymphoma stem cells in a murine ALCL-like model.

Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN AlkatrasFunder: DFG (CRC850)Funder: DFG (CRC850) BMBF (DeCaRe, FKZ 01ZX1409B)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras DFG (FOR 2033 B1)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras Grant from the Government of Baden-Württemberg (BSL)While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK+ lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4-/CD8- double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/β chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30+ mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease

Non-Invasive In Vivo Imaging of Tumor-Associated CD133/Prominin

detection of cancer stem cells is of great importance. detection of CD133/prominin, a cancer stem cell surface marker for a variety of tumor entities. The CD133-specific monoclonal antibody AC133.1 was used for quantitative fluorescence-based optical imaging of mouse xenograft models based on isogenic pairs of CD133 positive and negative cell lines. A first set consisted of wild-type U251 glioblastoma cells, which do not express CD133, and lentivirally transduced CD133-overexpressing U251 cells. A second set made use of HCT116 colon carcinoma cells, which uniformly express CD133 at levels comparable to primary glioblastoma stem cells, and a CD133-negative HCT116 derivative. Not surprisingly, visualization and quantification of CD133 in overexpressing U251 xenografts was successful; more importantly, however, significant differences were also found in matched HCT116 xenograft pairs, despite the lower CD133 expression levels. The binding of i.v.-injected AC133.1 antibodies to CD133 positive, but not negative, tumor cells isolated from xenografts was confirmed by flow cytometry. imaging of tumor-associated CD133 is feasible and that CD133 antibody-based tumor targeting is efficient. This should facilitate developing clinically applicable cancer stem cell imaging methods and CD133 antibody-based therapeutics

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

The human plasminogen activator inhibitor-1 gene.

This thesis focuses on the human gene for plasminogen activator inhibitor-1 (PAI-1). The generation of the active serine protease plasmin plays an important role in many biological processes including fibrinolysis, cell migration, tumor metastasis, tissue remodeling, and ovulation. Plasmin is first synthesized as the proenzyme plasminogen, which is activated by either tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). PAI-1 inhibits this activation via its interaction with both uPA and tPA. The first portion of the thesis describes the isolation and characterization of the structural gene for human PAI-1. This gene was found to span approximately 12 kb and contains eight introns. The start point of transcription was localized to 142 nucleotides upstream from the start codon. A segment containing 730 nucleotides of 5\sp\prime upstream region was shown to function as a promoter in COS cells. The second section of the dissertation is a detailed comparison of PAI-1 gene structure with other members of the serine protease inhibitor (serpin) gene family. Although the locations of the human and rat PAI-1 introns are identical, little conservation of intron positions was observed when compared to other serpins. It is proposed that intron location among gene family members may be a better predictor of relatedness than amino acid sequence. Finally, a novel method for quantitating RNA from small numbers of cells using the polymerase chain reaction (PCR) was developed in order to examine the regulation and expression of PAI-1. Using this procedure the previously observed decrease of PAI-1 mRNA levels in human umbilical vein endothelial cells (HUVEC) in the presence of ECGF and heparin was confirmed. An estimate for PAI-1 mRNA abundance of approximately 1700 molecules per cell was obtained for 6th passage HUVEC. Comparing PAI-1 mRNA levels in the nucleus with those present in the cytoplasm suggests that the effect of ECGF/heparin on mRNA level may occur at the nuclear rather than post-nuclear level. This approach may prove to be of general utility for the study of gene expression in a number of other instances.Ph.D.Human GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105367/1/9124007.pdfDescription of 9124007.pdf : Restricted to UM users only

Antimicrobial Photoinactivation of In Situ Oral Biofilms by Visible Light Plus Water-Filtered Infrared A and Tetrahydroporphyrin-tetratosylate (THPTS)

The aim of this study was to examine the effect of aPDT with visual light (VIS) + water-filtered infrared A (wIRA) as a light source, and tetrahydroporphyrin-tetratosylate (THPTS) as a photosensitizer on in situ initial and mature oral biofilms. The samples were incubated, ex situ, with THPTS for two minutes, followed by irradiation with 200 mW cm - 2 VIS + wIRA for five minutes at 37 °C. The adherent microorganisms were quantified, and the biofilm samples were visualized using live/dead staining and confocal laser scanning microscopy (CLSM). The THPTS-mediated aPDT resulted in significant decreases in both the initially adherent microorganisms and the microorganisms in the mature oral biofilms, in comparison to the untreated control samples (>99.99% each; p = 0.018 and p = 0.0066, respectively). The remaining vital bacteria significantly decreased in the aPDT-treated biofilms during initial adhesion (vitality rate 9.4% vs. 71.2% untreated control, 17.28% CHX). Of the mature biofilms, 25.67% remained vital after aPDT treatment (81.97% untreated control, 16.44% CHX). High permeability of THPTS into deep layers could be shown. The present results indicate that the microbial reduction in oral initial and mature oral biofilms resulting from aPDT with VIS + wIRA in combination with THPTS has significant potential for the treatment of oral biofilm-associated diseases