Stage-dependent localization of F-actin and Na+ /K+ -ATPase in zebrafish embryos detected using optimized cryosectioning immunostaining protocol.

The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.

Intercellular nuclear migration in cryofixed tobacco male meiocytes.

In this study, intercellular nuclear migration (INM), also known as cytomixis, was documented in cryofixed plant meiocytes for the first time. Intact tobacco inflorescences and flower buds as well as dissected individual anthers were cryofixed in liquid nitrogen by plunge freezing. Cryosubstituted and cryosectioned male meiocytes were analyzed by light microscopy. For cryosubstitution, the frozen material was kept in acetic alcohol at - 70 °C for 1 week. For cryosectioning, the frozen material was sectioned at - 20 °C, and fixed with precooled acetic alcohol. Fixation of the intact tobacco inflorescences in Carnoy's solution was used as a control. Microscopy revealed good preservation of cell structure in the cryofixed anthers, flower buds, and inflorescences. INM was detectable in all the studied cryofixed and chemically fixed samples. The cytological picture of INM observed in the cryofixed meiocytes did not noticeably differ from the picture obtained with the chemically fixed cells. These results indicate that INM is observable irrespective of whether a physical or chemical fixation method is employed, with minimal damage from handling. Our results contradict the notion that INM is a phenomenon caused by mechanical, osmotic, or chemical artifacts during sample preparation.

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes.

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss.

Feasibility study of cryobiopsy for practical pathological diagnosis of primary lung cancer including immunohistochemical assessment.

BACKGROUND: Precision medicine in non-small cell lung cancer requires attainment of a sufficient amount of high-quality tumor tissue. Transbronchial cryobiopsy has emerged as a new diagnostic method for non-neoplastic lung disease with a better potential to assess morphology compared with conventional methods. However, the influence of cryobiopsy on specimen quality, particularly detection of protein expression, is unknown. We performed a comparative immunohistochemical study in specimens obtained by cryobiopsy versus conventional sampling to evaluate the feasibility of cryobiopsy for lung cancer diagnosis. METHODS: Pairs of artificial biopsy specimens, collected using a cryoprobe or conventional scalpel, were obtained from 43 surgically resected primary lung tumors. Formalin-fixed, paraffin-embedded blocks were prepared in an ISO15189-certified laboratory. Immunohistochemical staining of thyroid transcription factor-1, p40, Ki67 and programmed death-ligand 1 (22C3) was performed. The H-scores for thyroid transcription factor-1 and p40, labeling index for Ki67 and tumor proportion score for programmed death-ligand 1 were assessed. Pearson's correlation coefficients between two sampling types were calculated. RESULTS: The thyroid transcription factor-1 and p40 H-scores showed perfect correlations between the cryobiopsy and conventional scalpel-obtained specimens (R2 = 0.977 and 0.996, respectively). Ki67 labeling index and PD-L1 tumor proportion score also showed strong correlations between the two sample types (R2 = 0.896 and 0.851, respectively). Five cases (11.6%) exhibited differences in tumor proportion score category between sample types, potentially because of intratumoral heterogeneity. CONCLUSIONS: Immunohistochemical expression of certain tumor markers showed a high concordance between cryobiopsy and conventional scalpel sampling. Cryobiopsy is feasible for pathological diagnostics including PD-L1 evaluation.

Protocol on Tissue Preparation and Measurement of Tumor Stiffness in Primary and Metastatic Colorectal Cancer Samples with an Atomic Force Microscope.

Tumors have complex microenvironments that provide not only biochemical but also mechanical cues to the cells within. In particular, tumor stiffness has been shown to regulate tumor growth, invasion, and metastasis. Understanding how the mechanical microenvironment controls cell behavior could be key to unraveling new therapeutic approaches. Here, we describe a protocol to prepare primary and metastatic human colorectal cancer samples and measure tumor stiffness at the tissue level using an atomic force microscope in spectroscopy mode. For complete details on the use and execution of this protocol, please refer to Shen et al. (2020).

Integrating High-Resolution MALDI Imaging into the Development Pipeline of Anti-Tuberculosis Drugs.

Successful treatment of tuberculosis (TB) requires antibiotics to reach their intended point of action, i.e., necrotizing granulomas in the lung. MALDI mass spectrometry imaging (MSI) is able to visualize the distribution of antibiotics in tissue, but resolving the small histological structures in mice, which are most commonly used in preclinical trials, requires high spatial resolution. We developed a MALDI MSI method to image antibiotics in the mouse lung with high mass resolution (240k @ m/z 200 fwhm) and high spatial resolution (10 µm pixel size). A crucial step was to develop a cryosectioning protocol that retains the distribution of water-soluble drugs in small and fragile murine lung lobes without inflation or embedding. Choice and application of matrices were optimized to detect human-equivalent drug concentrations in tissue, and measurement parameters were optimized to detect multiple drugs in a single tissue section. We succeeded in visualizing the distribution of all current first-line anti-TB drugs (pyrazinamide, rifampicin, ethambutol, isoniazid) and the second-line drugs moxifloxacin and clofazimine. Four of these compounds were imaged for the first time in the mouse lung. Accurate mass identification was confirmed by on-tissue MS/MS. Evaluation of fragmentation pathways revealed the structure of the double-protonated molecular ion of pyrazinamide. Clofazimine was imaged for the first time with 10 µm pixel size revealing clofazimine accumulation in lipid deposits around airways. In summary, we developed a platform to resolve the detailed histology in the murine lung and to reliably detect a range of anti-TB drugs at human-equivalent doses. Our workflow is currently being employed in preclinical mouse studies to evaluate the efficacy of novel anti-TB drugs.

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

Targeting Endothelin Receptors in a Murine Model of Myocardial Infarction Using a Small Molecular Fluorescent Probe.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time-Lapse Analysis of 3D Cultures.

When performing histology of softer biomaterials, aspiration disrupts the cellular and molecular location information. This study aims to develop a cryosectionable well insert able to preserve the biomaterial and cell's original 3D conformation from the well to histology analysis. The well insert is composed of a paraffin-coated gelatine pill. Within the coated capsule, the human epithelial cell line (NS-SV-AC) is cultured in Matrigel, GrowDex, Myogel, Myogel + GrowDex, or cell culture media for 14 days. At 0 and 14 days, the samples are frozen in liquid nitrogen and cryotome is used to create sections. The slides are stained by Sirius Red and immunohistochemistry using antibodies human collagens I-V and human Ki-67. Sirius Red shows pink shades of biomaterials and the best cellular vertical distribution throughout the sagittal section of the well is achieved with Matrigel, GrowDex, and Myogel + GrowDex; in Myogel and media, the cells sink. For collagen protein expression, only Matrigel induces a notable difference while in the other materials, collagen staining is weak or difficult to distinguish from endogenous collagens. Ki-67 expression is maintained over time. The 3D-cryo well insert provides a new time-lapse histology perspective of analysis for liquid or gel cultures that maintains cells and macromolecules in their unaltered in-well configuration.

A novel intraoral injection technique for rat levator veli palatini muscle regeneration.

BACKGROUND: The levator veli palatini (LVP) muscle drives the elevation and retraction of the soft palate to facilitate speech and feeding, but undergoes atrophic changes in patients with cleft palate deformity. This study aimed to establish an effective drug delivery technique for LVP muscle regeneration. METHODS: An intraoral injection technique for rat LVP muscle regeneration was developed based on careful examination of the rat craniofacial anatomy. The accuracy and reliability of this technique were tested by cone-beam computed tomography and nitrocellulose dye labeling. Recombinant human Wnt7a was delivered via this injection technique, and the subsequent responses of the levator veli palatini muscle were analyzed. RESULTS: Both the cone-beam computed tomography orientation of the needle tip and dye labeling suggested repeatable accuracy of the injection technique. Recombinant human Wnt7a delivery via this technique induced regeneration-related changes, including increased expression of centrally nucleated myofibers and Ki67+ve nuclei. CONCLUSION: The intraoral injection technique is safe and efficient. It can be used for accurate drug delivery and to screen regenerative therapeutics for the LVP muscle.

Mohs Micrographic Surgery.

Mohs micrographic surgery (MMS) is the gold standard for treating various cutaneous tumors. MMS has evolved into a single-day, outpatient procedure. The tumor is excised, mapped, and processed with frozen, horizontal sections for immediate histologic evaluation. The process is repeated as necessary until the tumor is completely removed, with maximal conservation of normal tissue. Evaluation of 100% of the surgical margin allows for exceptional cure rates. The Mohs surgeon is trained in tumor excision, histopathology interpretation, and surgical reconstruction. The use of MMS is often part of a multidisciplinary approach to treating cutaneous tumors.

Microanatomy Around the Facial Nerve Pathway for Microvascular Decompression Surgery Investigated with Correlative Light Microscopy and Block-Face Imaging.

BACKGROUND: Microvascular decompression for hemifacial spasm is performed at the root exit zone. More proximal segments of the facial nerve, defined as the root emerging zone (REmZ), may also be susceptible to neurovascular compression. Consequently, detailed knowledge of the microanatomy around facial nerve fibers at the pontomedullary junction is essential for consistent success of microvascular decompression. METHODS: Five human brainstems obtained from cadavers were investigated using correlative light microscopy and block-face imaging, which obtains arbitrary two-dimensional light microscopic and three-dimensional volume data from a single specimen. The entire facial nerve pathway, including the myelin transition, was evaluated inside and outside the brainstem. RESULTS: Correlative light microscopy and block-face imaging showed the intra-brainstem facial nerve fibers emerging at the brainstem surface deep at the pontomedullary sulcus (REmZ) and coursing along the pontine surface before detaching from the pons (root exit zone). An acute emerging angle significantly increased the surface area with central myelin. The facial nerve bundle formed 1 fasciculus in the portion covered by central myelin but divided into 2 fasciculi in the myelin transitional portion and then into multiple fasciculi more distally. Arteries around the REmZ were often anchored by perforating branches entangled with lower cranial nerves. CONCLUSIONS: Facial nerve fibers are susceptible to vascular compression on emerging onto the deep brainstem surface at the pontomedullary sulcus. The key procedure in microvascular decompression is full dissection of the lower cranial nerves down to the brainstem origin to explore both the root exit zone and the REmZ.

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Immediate Label-Free Ex Vivo Evaluation of Human Brain Tumor Biopsies With Confocal Reflectance Microscopy.

Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin-stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking.

Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation.

BACKGROUND: The pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana. METHODS: Here, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. RESULTS: Dozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone. CONCLUSIONS: The major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses.

Transbronchial lung cryobiopsy: Associated complications.

INTRODUCTION: Transbronchial lung cryobiopsy (TBC) has emerged as a diagnostic alternative to surgical lung biopsy in interstitial lung disease (ILD). Despite its less invasive nature, some associated complications have been described. OBJECTIVE: To evaluate complications of TBC and associated factors. METHODS: Prospective evaluation of all patients with ILD submitted to TBC in our centre. Clinicodemographic variables and factors associated to TBC complications were analyzed. The effect of the variables on the complication risk was evaluated by a logistic regression model. RESULTS: Ninety patients were included (mean age 60±13 years; 58.9% male). Twenty-two patients presented pneumothorax, 18 (81.8%) of which were treated with chest tube drainage [median air leak time: 1 day (IQR=2)]. Grade 2 and 3 bleeding was observed in 13 (14.4%) cases. Presence of visceral pleura in the sample accounted for almost more than 10 times the odds of pneumothorax (OR=9.59, 95% CI 2.95-31.17, p<0.001). Increased body mass index (BMI) was associated with bleeding (16% additional odds for each BMI unit increase (OR=1.16, 95% CI 1.01-1.34, p=0.049). CONCLUSION: The most frequent complication of TBC was pneumothorax, although rapidly reversible. There was a positive association between pneumothorax and the presence of pleura in the biopsy samples as well as between bleeding and increased BMI. More studies about TBC complications are needed to improve the selection of the candidates for this procedure.

Blood-Brain Barrier Penetrating Biologic TNF-α Inhibitor for Alzheimer's Disease.

Tumor necrosis factor alpha (TNF-α) driven processes are involved at multiple stages of Alzheimer's disease (AD) pathophysiology and disease progression. Biologic TNF-α inhibitors (TNFIs) are the most potent class of TNFIs but cannot be developed for AD since these macromolecules do not cross the blood-brain barrier (BBB). A BBB-penetrating TNFI was engineered by the fusion of the extracellular domain of the type II human TNF receptor (TNFR) to a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), designated as the cTfRMAb-TNFR fusion protein. The cTfRMAb domain functions as a molecular Trojan horse, binding to the mouse TfR and ferrying the biologic TNFI across the BBB via receptor-mediated transcytosis. The aim of the study was to examine the effect of this BBB-penetrating biologic TNFI in a mouse model of AD. Six-month-old APPswe, PSEN 1dE9 (APP/PS1) transgenic mice were treated with saline (n = 13), the cTfRMAb-TNFR fusion protein (n = 12), or etanercept (non-BBB-penetrating biologic TNFI; n = 11) 3 days per week intraperitoneally. After 12 weeks of treatment, recognition memory was assessed using the novel object recognition task, mice were sacrificed, and brains were assessed for amyloid beta (Aß) load, neuroinflammation, BBB damage, and cerebral microhemorrhages. The cTfRMAb-TNFR fusion protein caused a significant reduction in brain Aß burden (both Aß peptide and plaque), neuroinflammatory marker ICAM-1, and a BBB disruption marker, parenchymal IgG, and improved recognition memory in the APP/PS1 mice. Fusion protein treatment resulted in low antidrug-antibody formation with no signs of either immune reaction or cerebral microhemorrhage development with chronic 12-week treatment. Chronic treatment with the cTfRMAb-TNFR fusion protein, a BBB-penetrating biologic TNFI, offers therapeutic benefits by targeting Aß pathology, neuroinflammation, and BBB-disruption, overall improving recognition memory in a transgenic mouse model of AD.

How to obtain good morphology and antigen detection in the same tissue section?

Most human and animal biopsy samples are routinely embedded in paraffin since this enables the pathologist or researcher to obtain excellent morphology and simplifies storage. Nevertheless, in many cases, the antigen of interest cannot be detected in paraffin section. The alternative available for good immunohistochemistry is preparation of cryosections, which usually provide decent antigen preservation and are frequently used for immunofluorescence. However, cryosections often do not provide efficient morphological details of tissues and cells for pathologic evaluation. In order to obtain good antigen preservation and improve tissue and cell morphology after freezing, we tested three different fixations and freezing methodologies and compared them to routine formaldehyde fixation and paraffin embedding. As a model system, we selected the epithelium of the rat urinary bladder and trachea. On all samples, haematoxylin and eosin staining was performed as well as immunofluorescence with antibodies against tight junction protein ZO-1 and against intermediate filament cytokeratin 7. The best compromise between morphology and immunofluorescence was obtained with "sucrose impregnation prior to freezing" method. Moreover, this procedure is also quicker in comparison to standard paraffin section preparation. To check the clinical relevance of our study, this method was used for human biopsy samples of neoplastic urothelial and bronchial mucosa lesions. Besides good immunofluorescence results, the morphology of these samples was well preserved. We therefore propose that cryosection preparation with sucrose impregnation prior to freezing should be further exploited in other clinical and veterinary applications, since it enables good morphology and antigen preservation.

Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech.

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68+ and HmAIF-1+ macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

Tumorigenicity and Validity of Fluorescence Labelled Mesenchymal and Epithelial Human Oral Cancer Cell Lines in Nude Mice.

Tumorigenicity and metastatic activity can be visually monitored in cancer cells that were labelled with stable fluorescence. The aim was to establish and validate local and distant spread of subcutaneously previously injected fluorescence transduced human tongue cancer cell lines of epithelial and mesenchymal phenotype in nude mice. A total of 32 four-week-old male athymic Balb/c nude mice were randomly allocated into 4 groups (n = 8). A single dose of 0.3 mL PBS containing 1 × 107 of four different cancer cell-lines (UM1, UM1-GFP, UM2, and UM2-RFP) was injected subcutaneously into the right side of their posterolateral back. Validity assessment of the labelled cancer cells' tumorigenicity was assessed by physical examination, imaging, and histology four weeks after the injection. The tumor take rate of cancer cells was similar in animals injected with either parental or transduced cancer cells. Transduced cancer cells in mice were easily detectable in vivo and after cryosection using fluorescent imaging. UM1 cells showed increased tumor take rate and mean tumor volume, presenting with disorganized histopathological patterns. Fluorescence labelled epithelial and mesenchymal human tongue cancer cell lines do not change in tumorigenicity or cell phenotype after injection in vivo.