A Prospective Observational Study on the Role of Immunohistochemical Expression of Orphanin in Laryngeal Squamous Cell Carcinoma Recurrence.

To date, histological biomarkers expressed by laryngeal cancer are poorly known. The identification of biomarkers associated with laryngeal squamous cell carcinoma (SCC), would help explain the tumorogenesis and prevent the possible recurrence of the lesion after treatment. For this reason, the aim of this study is to investigate, for the first time, the Orphanin expression in 48 human specimens of laryngeal SCC and evaluate its possible correlation with patients prognosis. We analyzed pathological specimens from 48 patients with laryngeal SCC to detect the presence of Orphanin by using an immunohistochemistry test. We compared the findings with healthy tissue acquired from patients who underwent surgery for mesenchymal benign tumours of the larynx. The specimens were stained with anti-Orphanin monoclonal antibodies. Results were processed through a computerised image analysis system to determine a scale of staining intensity. All the tumoural specimens examined showed a significant immunoreaction for Orphanin when compared with healthy tissues (p < 0.05) but with a different immune reactivity related to clinical-pathological features. A high Orphanin expression was not significantly related to Histological Grading (HG), TNM, and stage (p > 0.05). In the multivariate analysis, the Orphanin expression was significantly related only to the malignant recurrence (p < 0.05). Our study suggests that Orphanin could have a role in tumorigenesis by increasing the recurrence of cancer; therefore, it should be further explored as a possible biomarker for laryngeal cancer.

COVID-19 Vaccine-Related Thrombosis: A Systematic Review and Exploratory Analysis.

Introduction: The World Health Organization declared the coronavirus disease 2019 (COVID-19) pandemic on March 11, 2020. Two vaccine types were developed using two different technologies: viral vectors and mRNA. Thrombosis is one of the most severe and atypical adverse effects of vaccines. This study aimed to analyze published cases of thrombosis after COVID-19 vaccinations to identify patients' features, potential pathophysiological mechanisms, timing of appearance of the adverse events, and other critical issues. Materials and Methods: We performed a systematic electronic search of scientific articles regarding COVID-19 vaccine-related thrombosis and its complications on the PubMed (MEDLINE) database and through manual searches. We selected 10 out of 50 articles from February 1 to May 5, 2021 and performed a descriptive analysis of the adverse events caused by the mRNA-based Pfizer and Moderna vaccines and the adenovirus-based AstraZeneca vaccine. Results: In the articles on the Pfizer and Moderna vaccines, the sample consisted of three male patients with age heterogeneity. The time from vaccination to admission was ≤3 days in all cases; all patients presented signs of petechiae/purpura at admission, with a low platelet count. In the studies on the AstraZeneca vaccine, the sample consisted of 58 individuals with a high age heterogeneity and a high female prevalence. Symptoms appeared around the ninth day, and headache was the most common symptom. The platelet count was below the lower limit of the normal range. All patients except one were positive for PF4 antibodies. The cerebral venous sinus was the most affected site. Death was the most prevalent outcome in all studies, except for one study in which most of the patients remained alive. Discussion: Vaccine-induced thrombotic thrombocytopenia (VITT) is an unknown nosological phenomenon secondary to inoculation with the COVID-19 vaccine. Several hypotheses have been formulated regarding its physiopathological mechanism. Recent studies have assumed a mechanism that is assimilable to heparin-induced thrombocytopenia, with protagonist antibodies against the PF4-polyanion complex. Viral DNA has a negative charge and can bind to PF4, causing VITT. New experimental studies have assumed that thrombosis is related to a soluble adenoviral protein spike variant, originating from splicing events, which cause important endothelial inflammatory events, and binding to endothelial cells expressing ACE2. Conclusion: Further studies are needed to better identify VITT's pathophysiological mechanisms and genetic, demographic, or clinical predisposition of high-risk patients, to investigate the correlation of VITT with the different vaccine types, and to test the significance of the findings.

Caspase 9 and Caspase 3 Immunohistochemical Pattern in Skeletal and Cardiac Muscles at Different Times after Death: An Experimental Study on PMI Estimation.

(1) Background: The estimation of the post mortem interval (PMI) is a challenge for forensic pathologists because data emerging from methods commonly applied are not always conclusive, since several conditions exist that may affect the reliability of these parameters. Thus, new approaches have been proposed to overcome such a limit. In recent years, several studies have been performed on proteins analyzing their expression/degradation patterns in relation to the progressing of the post mortem interval. (2) Methods: The immunoreactivity patterns of two apoptosis mediators-Caspase 9 and Caspase 3-have been tested in order to evaluate their potential role as markers of the post mortem interval. The immunohistochemical analysis was performed on samples of skeletal and cardiac muscles obtained from rats at 0, 4, 8, 12, 24 and 72 h after death. (3) Results: The observed immunoreactivity patterns of both Caspase 9 and Caspase 3 showed a significant correlation with increasing post mortem interval either in skeletal or cardiac muscles, while the comparison of the immunoreactivity patterns of the two apoptotic mediators within each tissue appeared consistent with a preliminary activation of the "initiator" Caspase 9, which, in turn, subsequently activates the "executioner" Caspase 3. (4) Conclusion: The different expressions and decrease immunohistochemically observed on both caspases with progressing PMI support the usefulness of the combined analysis for post mortem interval estimation.

Wharton's Jelly Mesenchymal Stromal Cells from Human Umbilical Cord: a Close-up on Immunomodulatory Molecules Featured In Situ and In Vitro.

Therapeutic options for end-stage organ failure are often limited to whole organ transplantation. The tolerance or rejection of the transplanted organ is driven by both early non-specific innate and specific adaptive responses. The use of mesenchymal stromal cells (MSCs) is considered a promising tool in regenerative medicine. Human umbilical cord (HUC) is an easily available source of MSCs, without relevant ethical issues. Moreover, Wharton's jelly-derived MSCs (WJ-MSCs), showed consistent immunomodulatory features that may be useful to promote immune tolerance in the host after transplantation. Few data are available on the phenotype of WJ-MSCs in situ. We investigated the expression of immune-related molecules, such as HLAs, IDO, CD276/B7-H3, and others, both in situ (HUC) and in in vitro-cultured WJ-MSCs. Morphological and biochemical techniques were used to define the expression of such molecules. In addition, we focused on the possible role of CD276/B7-H3 on T cells proliferation inhibition. We assessed CD276/B7-H3 expression by WJ-MSCs both in situ and alongside cell culture. WJ-MSCs were able to suppress T cell proliferation in mixed lymphocyte reaction (MLR). Moreover, we describe for the first time a specific role for CD276/B7-H3, since the immunomodulatory ability of WJ-MSCs was abolished upon anti-CD276/B7-H3 antibody addition to the MLR. These results further detail the immune regulation properties and tolerance induction exerted by human WJ-MSCs, in particular pointing to CD276/B7-H3 as one of the main involved factors. These data further suggest WJ-MSCs as potent tools to modulate local immune response in "support-type" regenerative medicine approaches.

A possible biomarker for methadone related deaths.

Methadone (MTH) concentrations in those dying of MTH toxicity totally overlap concentrations where the presence of MTH is only an incidental finding, making it very difficult to make distinctions in actual cases. A biomarker, be it anatomical or biochemical for MTH toxicity is badly needed, particularly if that markers were known to disrupt effective ventilation. Because the brainstem houses the regulatory centers for cardiorespiratory-control enters, it would seem to be the most likely anatomical site to seek abnormalities in cardiorespiratory control. OBJECTIVE: To locate and describe the cells of nucleus of the solitary tract (TS)(NTS) in human brainstem and determine if neuronal cell death, either necrotic or apoptotic, within the TS of humans is more common in deaths due directly to MTH toxicity than with in the solitary tract itself. DESIGN, SETTING, PARTICIPANTS: This was a single cohort study of MTH related decedents autopsied at a large university hospital. Each decedent had a recent history of non medical/illicit MTH use and had been pronounced dead in the field, prior to ever reaching the hospital. Complete autopsy and complete toxicology testing were performed on the formalin fixed brains of each individual. Multiple blocks were prepared of the area of interest, namely the tissue lying immediately between the inferior and the super colliculi. This volume, by definition, would have included the area of the Rostral Ventrolateral Medulla (RVLM), the location of the TS. Immunohistochemistry studies utilizing caspase-9 reaction (a protease enzyme involved in the process of preprogrammed death) were performed in order to estimate the degree and proportion of neuronal apoptosis, and also access the degree of classical necrosis within the NTS. MAIN OUTCOMES AND MEASURES: The primary outcome measure was the presence or absence of neuronal apoptosis and/or necrosis within the NTS. RESULTS: Cells displaying evidence of early apoptosis and advanced apoptosis, consisting primarily of nuclear fragmentation, admixed with other neurons displaying the features of classic necrosis were found. Evidence of classic necrosis was identifiable in most of the controls, though minor degrees of apoptosis were identifiable with Caspase staining and quantitative image analysis of immunohistochemical stains. CONCLUSIONS: and Relevance: Our study shows that neurons, primarily along the TS, but occasionally in other cell nuclei (even controls) are vulnerable, both to direct MTH toxicity (via apoptosis) and indirectly (via hypoxia leading to classical cell necrosis). When MTH is found to be present in significant concentrations, but apoptotic lesions are absent, it would be reasonable to assume that MTH was not primarily the cause of cardiorespiratory arrest.

Comparative analysis of Hsp10 and Hsp90 expression in healthy mucosa and adenocarcinoma of the large bowel.

BACKGROUND: Heat shock proteins (Hsps) assist other proteins in their folding and drive the degradation of defective proteins. During evolution, these proteins have also acquired other roles. Hsp10 is involved in immunomodulation and tumor progression. Hsp90 stabilizes a range of "client" proteins involved in cell signaling. The present study evaluated the expression levels of Hsp10 and Hsp90 in normal mucosa and adenocarcinoma samples of human large bowel. MATERIALS AND METHODS: Samples of normal mucosa and adenocarcinoma were collected and Reverse transcriptase-polymerase chain reaction RT-PCR, western blotting (WB) analyses, as well as immunohistochemistry were performed to evaluate the expression levels of Hsp10 and Hsp90. RESULTS: RT-PCR showed a higher gene expression of Hsp10 and Hsp90 in adenocarcinoma samples compared to healthy mucosa. WB results confirmed these findings. Immunohistochemistry revealed higher levels of Hsp10 in adenocarcinoma in both the epithelium and the lamina propria, while Hsp90 expression was higher in the adenocarcinoma samples only in the lamina propria. CONCLUSION: Hsp10 and Hsp90 may be involved in large bowel carcinogenesis.

Immunohistochemical expression and distribution of orexin, orphanin and leptin in the major salivary glands of some mammals.

The aim of the study was to determine by immunochemistry the expression of leptin, orexin A and orphanin FQ in the major salivary glands (parotid, submandibular and sublingual) of rat, sheep and cow. These peptides, originally synthesized in central nervous system, adipose tissue and peripheral tissues including gastrointestinal tract, play an orexigenic (orphanin and orexin) or anorexigenic (leptin) roles in the intricate neuronal network appointed to the control of nutritional homeostasis. Peptide-specific immunoreactivity was present in the studied salivary glands with various intensities in different species, in the ductal epithelium, sometimes in the acinar epithelium, and in nervous trunks spread in connective tissue stroma. The obtained data show that salivary glands present an unexpected source of orexigenic and anorexigenic peptides which with their autocrine, paracrine, and endocrine mechanisms of action may participate in the control of salivary gland function.

Immunohistochemical expression of apoptotic factors, cytokeratins, and metalloproteinase-9 in periapical and epithelialized gingival lesions.

The aim of the study was to assess the involvement of apoptotic factors, cytokeratins and metalloproteinase-9 in the histogenesis of both Epithelialized Gingival Lesions (EGL) and Periapical Lesions (PAL). 55 consecutive patients, 30 with PAL and 25 with EGL, were selected for the study after clinical and radiological examinations. The PAL patients had severe periapical lesions and tooth decay with exposure of the pulp chamber.All PAL and EGL biopsies were surgically extracted, fixed in 10% buffered formalin, and processed for routine light microscopy. Ten biopsies of each category were processed for immunohistochemistry (IHC). Serial paraffin sections were stained by IHC with appropriate antibodies to detect cytokeratins (CKs) 1, 5, 8, 10 and 14, caspase-3 and -9, metalloproteinase-9, and for PCNA and TUNEL assays. Both PAL and EGL showed a high expression of the cytokeratin 1, 5 and 8 with higher expression in EGL. Moreover, CK10 was markedly less intense expressed in EGL compared to PAL, while CK14 was almost three times stronger expressed in EGL. The expression of caspase-3 and -9 was stronger in PAL compared to EGL, however, the difference was only significant for caspase-9. In PAL apoptosis detected by TUNNEL method and the expression of MMP-9 were higher than in EGL, whereas PCNA was significantly more expressed in EGL. The results clearly suggest that both lesions have exclusively an epithelial origin and that epithelial proliferation was correlated with the degree of apoptosis in both entities. PAL and EGL presented mostly similar cytokeratin expression except for CK10 and CK14, though with marked differences in the distribution and intensity of IHC reactions. Finally, the degradation of extracellular matrix in both lesions could be partially attributed to the strong presence of MMP-9.