In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect ...H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.
In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 ...mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary to a sequence of the H. pylori 16S rRNA gene, was used. First, the potential cytotoxicity and genotoxicity of the probe was assessed by commercial assays. Further, the performance of the probe for detecting H. pylori at different pH conditions was tested in vitro, using fluorescence in situ hybridization (FISH). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy.
H. pylori SS1 strain infecting C57BL/6 mice was successfully detected by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy.
In the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo diagnosis of H. pylori infection using fluorescent LNA probes, which would be helpful to obtain an immediate diagnosis. Our results proved for the first time that FIVH method is applicable inside the body of a higher-order animal.
Chlorinated solvents including tetrachloroethene (perchloroethene and trichloroethene), are widely used industrial solvents. Improper use and disposal of these chemicals has led to a widespread ...contamination. Anaerobic treatment technologies that utilize Dehalococcoides spp. can be an effective tool to remediate these contaminated sites. Therefore, the aim of this study was to develop, optimize and validate peptide nucleic acid (PNA) probes for the detection of Dehalococcoides spp. in both pure and mixed cultures. PNA probes were designed by adapting previously published DNA probes targeting the region of the point mutations described for discriminating between the Dehalococcoides spp. strain CBDB1 and strain 195 lineages. Different fixation, hybridization and washing procedures were tested. The results indicated that the PNA probes hybridized specifically and with a high sensitivity to their corresponding lineages, and that the PNA probes developed during this work can be used in a duplex assay to distinguish between strain CBDB1 and strain 195 lineages, even in complex mixed cultures. This work demonstrates the effectiveness of using PNA fluorescence in situ hybridization to distinguish between two metabolically and genetically distinct Dehalococcoides strains, and they can have strong implications in the monitoring and differentiation of Dehalococcoides populations in laboratory cultures and at contaminated sites.
Lymphatic vessels are essential for skin fluid homeostasis and immune cell trafficking. Whether the lymphatic vasculature is associated with hair follicle regeneration is, however, unknown. Here, ...using steady and live imaging approaches in mouse skin, we show that lymphatic vessels distribute to the anterior permanent region of individual hair follicles, starting from development through all cycle stages and interconnecting neighboring follicles at the bulge level, in a stem cell‐dependent manner. Lymphatic vessels further connect hair follicles in triads and dynamically flow across the skin. At the onset of the physiological stem cell activation, or upon pharmacological or genetic induction of hair follicle growth, lymphatic vessels transiently expand their caliber suggesting an increased tissue drainage capacity. Interestingly, the physiological caliber increase is associated with a distinct gene expression correlated with lymphatic vessel reorganization. Using mouse genetics, we show that lymphatic vessel depletion blocks hair follicle growth. Our findings point toward the lymphatic vasculature being important for hair follicle development, cycling, and organization, and define lymphatic vessels as stem cell niche components, coordinating connections at tissue‐level, thus provide insight into their functional contribution to skin regeneration.
Synopsis
Like blood vasculature, lymphangiogenesis may have an impact on epithelial tissue development. Combined morphological profiling and genetic analyses now show structural and functional association between lymph vessels (LV) and the hair follicles (HF), providing new insights into principles of niche organization during skin regeneration and maintenance.
LVs in the mouse back skin associate with HFs in a polarized manner, interconnecting HF triads throughout the hair cycle.
Depletion of Wnt ligands in HF stem cells disrupts LV‐HF association.
LVs transiently increase their caliber at the onset of stem cell activation showing a distinct molecular signature.
Genetic ablation of LVs leads to premature exit from HF growth.
Lymph vessels and the hair follicles are structurally and functionally associated during skin growth and maintenance.
Background and Objectives
Keratinocyte carcinoma (KC) is the most common cancer worldwide, and squamous cell carcinoma (SCC) is the second most frequent subtype. Ablative fractional laser ...(AFL)‐assisted drug delivery significantly enhances the uptake of topically applied drugs. The objective of this study was to assess tumor response and perform a descriptive characterization of the local recruitment of immune cells and systemic immune mediator levels in an ultraviolet radiation (UVR)‐induced murine SCC model after AFL treatment alone and combined with topical imiquimod.
Study Design/Materials and Methods
Immunocompetent hairless mice (C3·Cg/TifBomTac, n = 74) were irradiated with solar‐simulated UVR until 3‐mm SCCs developed. The mice were divided into four interventional groups: AFL alone, AFL + imiquimod, imiquimod alone, and untreated SCC controls. AFL was given as a single treatment, whereas imiquimod was applied daily until the mice were euthanized on Days 0, 2, 7, or 14. SCCs were photographed and measured (mm) to assess the therapeutic response. Skin samples were processed for histopathological and immunohistochemical analyses, as well as for flow cytometry. Cytokine expression changes in sera were analyzed using ELISpot cytokine arrays.
Results
Treatment of mouse SCCs with AFL + imiquimod induced the most robust immune cell infiltration and the greatest proportion of tumor clearance compared to other interventions. Early innate immune cell infiltration was induced by AFL + imiquimod treatment as the number of neutrophils and macrophages had increased fourfold within 2 days of treatment initiation compared with untreated SCC control mice (P < 0.05). AFL treatment alone had a more limited effect, with a fourfold increase in neutrophils (P < 0.05) but no significant increase in the number of macrophages. Correspondingly, treatment with AFL + imiquimod had the greatest effects on the adaptive immune cell recruitment: CD4+ T‐helper cells increased threefold at Day 7 compared with untreated SCCs (P = 0.0001) and, notably, cytotoxic CD8+ T cells increased 14‐fold at Day 14 (P = 0.0112). In addition, FOXP3+ regulatory T cells (Tregs) increased 14‐fold at Day 7 (P = 0.0026), suggesting the resolution of the inflammatory infiltration. AFL treatment alone induced a moderate immune cell infiltration (a twofold increase in CD4+ T‐helper cells, P = 0.0200; a threefold increase in CD8+ T cells, P = 0.0100; and a 14‐fold increase in FOXP3+ Tregs at Day 14, P = 0.0021), whereas imiquimod alone did not significantly increase cell counts. AFL + imiquimod treatment increased CXCL12 serum levels threefold at Day 14 (P = 0.0200).
Conclusion
AFL treatment alone and in combination with imiquimod induces substantial tumor clearance associated with local recruitment of innate and adaptive immune cells in UVR‐induced murine SCCs. These results may provide a basis for new immunotherapeutic approaches to KC treatment.
•Normally erythropoietic protoporphyria (EPP) is an inherited photodermatosis.•In very rare cases EPP is acquired in association with a myeloid neoplasm.•We here report a case of acquired EPP who ...debuted with severe skin photosensitivity.•After onset of photosensitivity the case was diagnosed with a hematological disease.
Erythropoietic protoporphyria (EPP) is a rare genetic photodermatosis caused by loss-of-function mutations in the gene for ferrochelatase leading to accumulation of the fluorescent protoporphyrin IX (PpIX) in erythrocytes. The mutations are most often inherited mutations present in all cells causing inherited EPP. In very rare cases EPP are acquired in association with myelodysplastic syndromes or myeloproliferative neoplasms, conditions with genetic instability.
We report a case of acquired EPP in association with hematological disease. We followed erythrocyte PpIX concentration over a year and measured PpIX fluorescence in individual erythrocytes in a blood sample from the case using flow cytometry. The major proportion of erythrocytes did not fluoresce (84%), whereas 13% contained low PpIX fluorescence, 1% contained medium fluorescence, and 2% contained high fluorescence.
Our observation of the very skewed PpIX distribution in erythrocytes supports the description that acquired EPP is caused by a somatic mutation effecting a clone of hematopoietic cells.
Introduction In this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting ...the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary to a sequence of the H. pylori 16S rRNA gene, was used. First, the potential cytotoxicity and genotoxicity of the probe was assessed by commercial assays. Further, the performance of the probe for detecting H. pylori at different pH conditions was tested in vitro, using fluorescence in situ hybridization (FISH). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy. Results H. pylori SS1 strain infecting C57BL/6 mice was successfully detected by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy. Conclusions In the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo diagnosis of H. pylori infection using fluorescent LNA probes, which would be helpful to obtain an immediate diagnosis. Our results proved for the first time that FIVH method is applicable inside the body of a higher-order animal.
The prostate cancer gene 3 (PCA3) is a prostate-specific, non-protein-coding RNA. It is overexpressed in prostate cancer compared with the normal prostate and has a negative expression in other ...tissues. This case-control study sought to analyze the frequency of the polymorphism PCA3 -845 G>A in participants without prostate cancer and patients with metastatic prostate cancer.
Carriers of GA and AA genotype had a higher risk for metastatic prostate cancer (odds ratio OR for genotype GA, 1.79 95% confidence interval (CI), 1.14-2.29; p=0.007; OR for genotype AA, 2.38 95% CI, 1.22-4.65; p=0.006). Furthermore, the recessive model showed that A allele carriers have an increased risk for developing metastatic prostate cancer (OR, 1.91 95% CI, 1.26-2.90; p=0.001).
These results suggest a link between PCA3 and metastatic prostate cancer. The evaluation of individual genetic profiles, according to the PCA3 -845 G>A polymorphism, may elucidate the function of this gene and the mechanisms involved in its regulation and role in prostate cancer.
The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and ...microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2'-O-methyl RNAs (2'OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 degree C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others.
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