Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo.

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.

Lymph Vessels Associate with Cancer Stem Cells from Initiation to Malignant Stages of Squamous Cell Carcinoma.

Tumor-associated lymph vessels and lymph node involvement are critical staging criteria in several cancers. In skin squamous cell carcinoma, lymph vessels play a role in cancer development and metastatic spread. However, their relationship with the cancer stem cell niche at early tumor stages remains unclear. To address this gap, we studied the lymph vessel localization at the cancer stem cell niche and observed an association from benign skin lesions to malignant stages of skin squamous cell carcinoma. By co-culturing lymphatic endothelial cells with cancer cell lines representing the initiation and promotion stages, and conducting RNA profiling, we observed a reciprocal induction of cell adhesion, immunity regulation, and vessel remodeling genes, suggesting dynamic interactions between lymphatic and cancer cells. Additionally, imaging analyses of the cultured cells revealed the establishment of heterotypic contacts between cancer cells and lymph endothelial cells, potentially contributing to the observed distribution and maintenance at the cancer stem cell niche, inducing downstream cellular responses. Our data provide evidence for an association of lymph vessels from the early stages of skin squamous cell carcinoma development, opening new avenues for better comprehending their involvement in cancer progression.

Tumor Clearance and Immune Cell Recruitment in UV-Induced Murine Squamous Cell Carcinoma Exposed to Ablative Fractional Laser and Imiquimod Treatment.

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.

Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models.

The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.

Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

Nonsurgical Gluteal Volume Correction with Hyaluronic Acid: A Retrospective Study to Assess Long-term Safety and Efficacy.

Background: Augmentation and reshaping of body volume, particularly in the gluteal area, presents a significant challenge in aesthetic surgery. Hyaluronic acid (HA) fillers have emerged as an effective and safe tool for such indications, but literature examining nonsurgical gluteal reshaping with HA remains limited. This study aims to evaluate the long-term safety of using recommended volumes of HA body fillers for nonsurgical gluteal augmentation. Methods: A retrospective, observational study was carried out across multiple centers in Italy and the United Arab Emirates. The study involved participants between 22 and 53 years of age who underwent gluteal augmentation using HA body filler (HYAcorp MLF1/2) between 2017 and 2021, with up to 4 years and 7 months of follow-up. Participants and investigators independently evaluated the procedure's effectiveness by comparing pre- and posttreatment photographs. The Global Aesthetic Improvement Scale was used to assess posttreatment satisfaction by both participants and investigators. All adverse effects (AEs) were recorded. Results: The study included a diverse group of 91 participants. No serious adverse events were reported, with the majority of AE occurring shortly after treatment and resolving in 1 week. AEs were more frequently observed in participants with previous treatments using different substances in the treatment area. Conclusions: The real-world application of HA body filler (HYAcorp MLF1/2) for gluteal augmentation in the participants of this study showed the treatment's effectiveness, with no severe adverse events reported among the participants. High levels of satisfaction were reported among both participants and investigators.

Distribution of protoporphyrin IX in erythrocytes in a case of acquired erythropoietic protoporphyria.

BACKGROUND: 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. CASE REPORT: 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. DISCUSSION: 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.

Isolation of Cancer Stem Cells from Squamous Cell Carcinoma.

Different cancer stem cell (CSC) populations can be found in many types of cancer, including squamous cell carcinoma (SSC). Diverse reports showed that CSC play a crucial role in the relapse of different types of cancer. CSC sustains tumor growth due to their capacity to self-renew and their potential to initiate secondary tumors with metastatic cancer features. Therefore, the development of methods for the isolation of CSC is a key step to explore the mechanisms underlying CSC maintenance. In this chapter, we provide a method for isolating CSC from cutaneous SSC using immunofluorescence labeling to allow the specific purification of CSC by fluorescence-activated cell sorting (FACS). This method is based on the use of CSC membrane markers, allowing as well the isolation CSC from different mouse strains.

Heterocellular cadherin connections: coordinating adhesive cues in homeostasis and cancer.

This short insight covers some of the recent topics relevant to the field of cadherin-catenin adhesion in mediating connections between different cell types, so-called heterotypic or heterocellular connections, in both homeostasis and cancer. These scientific discoveries are increasing our understanding of how multiple cells residing in complex tissues can be instructed by cadherin adhesion receptors to regulate tissue architecture and function and how these cadherin-mediated heterocellular connections spur tumor growth and the acquisition of malignant characteristics in tumor cells. Overall, the findings that have emerged over the past few years are elucidating the complexity of the functional roles of the cadherin-catenin complexes. Future exciting research lies ahead in order to understand the physical basis of these heterotypic interactions and their influence on the behavior of heterogeneous cellular populations as well as their roles in mediating phenotypic and genetic changes as cells evolve through complex environments during morphogenesis and cancer.

Detection of Helicobacter pylori in the Gastric Mucosa by Fluorescence In Vivo Hybridization.

In this chapter, we describe a fluorescence in vivo hybridization (FIVH) protocol, using nucleic acid probes, for the detection of the bacterium Helicobacter pylori in the gastric mucosa of an infected C57BL/6 mouse model. This protocol should be easily extended to other microorganisms not only as a way to identify in vivo important microorganisms and their patterns of distribution within specific or at different anatomic sites, but also to better understand interaction mechanisms involving the microbiome and the human body.

Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model.

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.

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes.

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.

Effect of Native Gastric Mucus on in vivo Hybridization Therapies Directed at Helicobacter pylori.

Helicobacter pylori infects more than 50% of the worldwide population. It is mostly found deep in the gastric mucus lining of the stomach, being a major cause of peptic ulcers and gastric adenocarcinoma. To face the increasing resistance of H. pylori to antibiotics, antimicrobial nucleic acid mimics are a promising alternative. In particular, locked nucleic acids (LNA)/2'-OMethyl RNA (2'OMe) have shown to specifically target H. pylori, as evidenced by in situ hybridization. The success of in vivo hybridization depends on the ability of these nucleic acids to penetrate the major physical barriers-the highly viscoelastic gastric mucus and the bacterial cell envelope. We found that LNA/2'OMe is capable of diffusing rapidly through native, undiluted, gastric mucus isolated from porcine stomachs, without degradation. Moreover, although LNA/2'OMe hybridization was still successful without permeabilization and fixation of the bacteria, which is normally part of in vitro studies, the ability of LNA/2'OMe to efficiently hybridize with H. pylori was hampered by the presence of mucus. Future research should focus on developing nanocarriers that shield LNA/2'OMe from components in the gastric mucus, while remaining capable of diffusing through the mucus and delivering these nucleic acid mimics directly into the bacteria.

Detection of Dehalococcoides spp. by peptide nucleic acid fluorescent in situ hybridization.

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.

Hybridization-based detection of Helicobacter pylori at human body temperature using advanced locked nucleic acid (LNA) probes.

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 °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.

Molecular study of the PCA3 gene: genotypic analysis of PCA3 polymorphism -845G>A and metastatic prostate cancer.

AIMS: 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. RESULTS: 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). CONCLUSIONS: 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.