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Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle’s principle, “The whole is greater than the sum of its parts”, multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available.

Recent scientific investigations have reported a number of essential oils to interfere with intracellular signalling pathways and to induce apoptosis in different cancer cell types. In this paper, Lavandin Essential Oil (LEO), a natural sterile hybrid obtained by cross-breeding L. angustifolia × L. latifolia, was tested on human leukaemia cells (HL60). Based on the MTT results, the reduced cell viability of HL60 cells was further investigated to determine whether cell death was related to the apoptotic process. HL60 cells treated for 24 h with LEO were processed by flow cytometry, and the presence of Annexin V was measured. The activation of caspases-3 was evaluated by western blot and immunofluorescence techniques. Treated cells were also examined by scanning and transmission electron microscopy to establish the possible occurrence of morphological alterations during the apoptotic process. LEO main compounds, such as linalool, linalyl acetate, 1,8-cineole, and terpinen-4-ol, were also investigated by MTT and flow cytometry analysis. The set of obtained results showed that LEO treatments induced apoptosis in a dose-dependent, but not time-dependent, manner on HL60 cells, while among LEO main compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis.

Background/Objectives: Although melasma is highly prevalent, its pathogenesis is not yet fully understood. In the skin, endothelin-1 (ET-1) is primarily produced by keratinocytes in response to UVB exposure, which is mediated by an increase in IL- 1[alpha] or reactive oxygen species. ET-1 plays a role in melanogenesis by binding to specific receptor B (ERB) or receptor A (ERA). However, the expression of ET1, ERA, and ERB in melasma has not been systematically investigated. The objective of this study was to evaluate the expression of ET-1, ERA, and ERB in facial melasma compared to the adjacent unaffected skin. Methods: Cross-sectional study, with 40 skin samples (20: facial melasma; 20: adjacent unaffected skin) from women with facial melasma without treatment for 30 days except for sunscreen. A triple staining immunofluorescence technique was performed for anti-vimentin, DAPI, plus one of the following antibodies: (a) anti-ET1, (b) anti-ERA; (c) anti-ERB. Interfollicular areas on the slides of each topography (melasma; unaffected skin) were photographed in triplicate under confocal laser microscopy. The mean staining intensities of the image histograms (0-255 pixels intensity) were estimated for different types of cells (suprabasal keratinocytes, basal layer, and upper dermis) and were blindly compared between topographies. Results: The mean (SD) age of the participants was 44.9 (9.2). The expression of ET-1 was increased in the whole epidermis with melasma when compared to the adjacent skin, being 32.8% (CI95% 14.7%- 52.6%) higher in the spinous layer (p=0.013), 30.4% (CI95% 13.7%-47.9%) higher in the basal layer (p=0.014), and 29.7% (CI95% 11.4%-49.7%) higher in the melanocytes (p=0.006). There was no noticeable expression of ET-1 within the cells on the upper dermis. Neither ERA nor ERB resulted in differential epidermal expression between melasma and unaffected skin (p>0.1). Conclusion: ET-1 is expressed more intensely on the epidermis from the skin with facial melasma compared to the unaffected adjacent skin. Keywords: melasma, immunofluorescence technique, endothelin-1, endothelin receptor-A, endothelin receptor-B

Bisphenol A (BPA) is an extremely common polymer that is used in typical everyday products throughout the world, especially in food and beverage containers. Within the last ten years, it has been found that the BPA monomer tends to leach into foodstuffs, and nanogram concentrations of it may cause a variety of deleterious health effects. These health problems are very evident in developing children and in young adults. The aim of this study was to expose developing pigs to dietary BPA at both legally acceptable and ten-fold higher levels. Livers that had been exposed to BPA showed vacuolar degeneration, sinusoidal dilatation, vascular congestion and glycogen depletion that increased with exposure levels. Furthermore, the livers of these models were then examined for irregularities and double-labeled immunofluorescence was used to check the innervated hepatic samples for varying neuronal expression of selected neuronal markers in the parasympathetic nervous system (PSNS). It was found that both the PSNS and all of the neuronal markers showed increased expression, with some of them being significant even at recommended safe exposure levels. The implications are quite serious since these effects have been observed at recommended safe levels with expression increasing in-line with exposure levels. The increased neuronal markers studied here have been previously correlated with behavioral/psychological disorders of children and young adults, as well as with childhood obesity and diabetes. However, further research must be performed in order to develop a mechanism for the above-mentioned correlations.

Background A relationship between IgA nephropathy (IgAN) and celiac disease (CD) has been reported. We show the pathogenetic link for the first time. Case presentation A 39-year-old man with cystic fibrosis (CF) and CF-related diabetes started to present gross hematuria, back pain and headache. At admission, laboratory analysis showed increase in serum creatinine of 1.5 mg/dl, together with hematuria and mild proteinuria (1 g/24 h). He underwent a renal biopsy to investigate the cause of hematuria and renal failure. Biopsy was consistent with IgAN. In view of patient reported dyspepsia, an upper gastrointestinal endoscopy with duodenal biopsies was undertaken and was normal. We looked for mucosal deposits of tTG-2 in the duodenum and the renal mesangium. tTG-2 deposits were found both in the duodenum and in renal biopsies, where they topographically replicated mesangial IgA deposits. After one year on a continued gluten containing diet, the patient developed a Marsh 2 type duodenal pathology. Conclusions Our findings suggest a connection between CD and IgAN in terms of an immune-mediated gluten-induced pathogenesis even in the absence of villous atrophy and serum celiac autoantibodies.

Zinc transporter 3 (ZnT3) is one of the zinc transporters family. It is closely connected to the nervous system, where enables the transport of zinc ions from the cytoplasm to synaptic vesicles. This substance has been described within the central and peripheral nervous system, especially in the enteric nervous system (ENS). The aim of the present study was to describe the co-localization of ZnT3 with selected neuromediators and/or neuromodulators participating in sensory stimuli conduction in neurons of the ENS within the porcine esophagus. Co-localization of ZnT3 with substance P (SP), leucine enkephalin (LENK) and calcitonin gene-related peptide (CGRP) was studied using standard double-immunofluorescence technique. The obtained results show that ZnT3, SP and/or LENK may occur in the same enteric neurons, and the degree of co-localization of these substances clearly depends on the fragment of esophagus studied and the type of enteric ganglia. In contrast, the co-localization of ZnT3 with CGRP was not observed during the present investigation. The obtained results suggest that ZnT3 in the ENS may be involved in the conduction of sensory and/or pain stimuli.

Bisphenol A (BPA) is an endocrine-disrupting compound (EDC) that can be found nearly everywhere in our polluted world. BPA has been correlated with pathophysiologies that include psychological disorders, especially in children. This study used juvenile porcine models to investigate the effects of BPA on the liver of developing vertebrates in order to determine the effects of BPA on innervated hepatic samples with the use of double-labeled immunofluorescence. This study specifically investigated the sympathetic nervous system (SNS) colocalized with a specific neural marker (PACAP) that has previously been correlated with specific pathophysiologies in the literature. In the liver, it was observed that there were significantly increased nerve fibers in the SNS colocalized with the neural marker PACAP after exposure to BPA at concentrations considered to be “safe” with the same or more profound effects at higher exposure levels. The implications of childhood exposure to BPA are then discussed with regard to several correlation studies that have linked BPA exposure to behavioral/psychological disorders. It is possible that BPA exposure in childhood may upregulate the SNS and PACAP levels, thereby contributing to the correlations in the literature.

Aim: The aim of this study was to analyze the expression level and localization of Toll-like receptor (TLR) 4 in gingival samples of healthy and chronic periodontitis subjects by indirect immunofluorescence technique (IFT). Materials and Methods: In this study, gingival tissue samples were obtained from 25 healthy and 25 periodontitis individuals. The tissues were processed and the initial characterization was done by hematoxylin and eosin staining. The expression and localization of the TLR4 receptor were determined in the epithelial and connective layer cells of the gingival tissue using the indirect IFT. Immunofluorescence images were acquired and quantitative expression of TLRs was analyzed by calculating the percentage of cells showing positive results. Results: We found that the healthy control group exhibited significantly lower values of TLR4 expression in comparison with the periodontitis patients. We also found that in patients with periodontitis the concentration of TLR4 was higher in the epithelium as compared to their expression in connective tissue cells. Conclusions: These data suggested a definite involvement of TLR4 in initiating and progression of an inflammatory response in periodontitis.

Background: Previous studies have shown considerable variation in diagnostic performance of enzyme linked immunosorbent assays (ELISAs) for measuring antineutrophil cytoplasmic antibodies (ANCA) specific for proteinase 3 (PR3) and myeloperoxidase (MPO). Objective: To analyse the performance characteristics of different commercially available direct ANCA ELISA kits. Methods: ELISA kits for detecting PR3-ANCA and MPO-ANCA from 11 manufacturers were evaluated. Serum samples were taken from patients with Wegener’s granulomatosis (15), microscopic polyangiitis (15), other vasculitides (10), and controls (40). Results were compared with data obtained by indirect immunofluorescence (IFT). The diagnostic performance of the tests was analysed and compared by receiver operating characteristic (ROC) curve analysis. Results: Applying the manufacturers’ cut off resulted in great variation in sensitivity of the commercial PR3-ANCA kits for diagnosing Wegener’s granulomatosis (ranging from 13.3% to 66.7%), and of the MPO-ANCA kits for diagnosing microscopic polyangiitis (ranging from 26.7% to 66.7%). Specificities were relatively constant (from 96.0% to 100%). IFT was superior to all ELISAs (C-ANCA for Wegener’s granulomatosis: sensitivity 73.3%, specificity 98%; P-ANCA for microscopic polyangiitis: sensitivity 86.7%, specificity 98%). The sensitivities of PR3-ANCA and MPO-ANCA ELISA kits were increased by lowering the cut off values. This reduced specificity but increased overall diagnostic performance. Conclusions: The low sensitivity of some commercial kits reflects the high cut off levels recommended rather than methodological problems with the assays. Comparative analyses using sera from well characterised patients may help identify optimum cut off levels of commercial ANCA ELISA tests, resulting in better comparability of results among assays from different manufacturers.

Chronic ulcerative stomatitis (CUS) is a poorly understood disease with clinical and histologic overlap with lichen planus (LP). Unlike classic LP, direct immunofluorescence (DIF) studies in cases of CUS exhibit a granular pattern of IgG in nuclei of basal and parabasal cells. This study assesses the demographic, clinical, histologic, and DIF features of CUS. It is important to differentiate CUS from LP and other vesiculobullous diseases (VBD) because lesions of CUS are resistant to steroid therapy, which is typically used to control LP and VBD. A literature review and IRB-approved retrospective search of CUS was performed within the archives of the University of Florida (UF) Oral Pathology Biopsy Service from 2007 to 2017. Fifty-two cases were identified from the literature and seventeen new cases were identified in our series. All UF patients were female and the median age was 64-years. The majority of patients were Caucasian and the most common location was buccal mucosa. Frequent clinical presentations were pain, erythema, leukoplakia, and ulcerations. Histologic features included epithelial separation, atrophic epithelium, and a chronic inflammatory infiltrate. All cases were confirmed with DIF testing that showed a speckled pattern of IgG staining in basal and parabasal cell nuclei. Fibrinogen was present in eleven cases and two cases were positive for C3. The results of our series are in accordance with the literature. Since CUS has overlapping features with LP and VBD, clinicians and pathologists should consider this entity and confirm diagnosis with DIF testing when recalcitrant oral ulcerative diseases are encountered.