Predictive Value of Anti- E6 Oncoprotein (High Risk- Human Papilloma Virus) and p16 Ink4a for Detecting HPV in Oral Epithelial Dysplasia.

AIM: The aim of this study was to evaluate the expression of human papillomavirus (HPV) in oral potentially malignant disorders (OPMD) and to examine the association of HPV in histological grades of dysplasia using p16 and Anti-E6 oncoprotein immunohistochemistry (IHC). SUBJECTS AND METHODS: This study focused on clinically diagnosed oral potentially malignant disorders. Clinical parameters such as age, gender, habits, occupation, duration, site, and the type of the lesions were examined and the incisional biopsy was done on the selected cases for the histopathological diagnosis. Selected cases of OPMDs were screened immunohistochemically for HPV 16 and HPV 18 (high-risk group) positivity using p16INK4a and Anti-E6 oncoprotein. The immunohistochemical p16 expression was evaluated based on (a) percentage of p16 positive cases and (b) pattern of p16 staining in various grades of OPMD. RESULTS: Anti-E6 oncoprotein (HR-HPV) expression level was only detected in 11 cases (37%), and positive expression of p16 was found in three cases (10%), with variation in cell proportion and intensity. Subsequently, the association between p16 expression level and clinicopathological characteristic factors was analyzed and a significant association was found between age and histopathology. CONCLUSION: There was an association between HPV and OPMD. Both biomarker tests, HPV E6 and p16 immunocytochemistry had a specific role in the detection of HR-HPV. Anti-E6 immunocytochemistry can be a valuable test with higher specificity for HPV DNA detection in oral epithelial dysplasia without losing sensitivity.

Expression sites of immunohistochemistry markers in oral diseases - A scoping review.

Introduction: Immunohistochemistry (IHC) has not always been an easy field for the research beginners like postgraduates, research fellows and scientists. Meaningful interpretation of IHC positivity needs expertise. This could be made easier for beginners by developing a conceptual framework of markers. The literature review revealed a lack of qualitative evidence on the hitherto IHC studies on oral diseases about the overall expression of IHC markers and its comparison with pathology and normal tissues. Aim: This scoping review aimed to examine the literature and classify the various immunohistochemistry markers of oral diseases based on the tissue, cell and site of expression. Materials and Methods: The review was in accordance with Preferred Reporting Items for scoping reviews (PRISMA -ScR). Electronic databases such as PubMed and Cochrane were searched for relevant articles till 2021. Results: We included 43 articles. We found five different possibilities of the site of expression of a marker in a cell. They are the nucleus, cytoplasm, cell membrane, extracellular matrix or any of the above combinations. Based on the tissue of expression, we also mapped the markers expressed in oral diseases to their tissue of origin as ectoderm, endoderm, mesoderm and markers with multiple tissues of expression. Based on our results, we derived two classifications that give an overview of the expression of IHC markers in oral diseases. Conclusion: This scoping review derived new insight into the classification of IHC markers based on cell lineage, tissue and site of expression. This would enable a beginner to better understand a marker with its application and the interpretation of the staining in research. This could also serve as a beginner's guide for any researcher to thrive and explore the IHC world.

Fibroblastic Growth Factor as a Diagnostic and Prognostic Marker in Odontogenic Cysts and Tumors: A Systematic Review.

Cellular signaling proteins maintain the basic activities of cell and communication, between the cells for normal growth and development and pathological situation as well. Fibroblast growth factor (FGF) and fibroblast growth factor receptors (FGFRs) have a comparatively huge part to play in the cellular communication processes. Human FGF has 22 members, 18 ligands, and 4 tyrosine kinase receptors for binding and is expressed in a wide range of cells. Any alteration in these factors would disrupt their normal function, leading to various abnormalities. The aim of this systematic analysis, is to understand the FGFs, the physiological and pathological role of FGF in oral diseases, and to predict the use of FGF in the predilection toward odontogenic cyst and tumors. This review helps confer the role of FGF in various physiological and pathological aspects in systemic diseases and analyzes its role in diagnosis and prognosis of odontogenic cysts and tumors.

Clinico-Cytological Study of Fungal Elements in the Buccal Smears of Prediabetic, Diabetic, and Nondiabetic Individuals in Association with Burning Sensation.

INTRODUCTION: Diabetes mellitus is a metabolic disorder that may have profound effects on oral mucosa being prone to infections, particularly candidiasis. In healthy individuals, the candida is a commensal, but in diabetic patients, it forms severe colonization, even in the absence of any clinically evident lesion and may cause a burning sensation of the mouth. This study aims to identify the presence or absence of fungal elements in buccal smears of nondiabetic, prediabetic, and diabetic individuals and to correlate the presence or absence of burning sensation in the oral cavity. MATERIALS AND METHODS: A total of ninety individuals were selected for the study and divided into Group 1 - thirty cases of diabetics, Group 2 - thirty cases of prediabetic patients, and Group 3 - thirty cases of nondiabetic individuals. Detailed case history and thorough intra and extraoral examination were recorded. The buccal smears were cultured in Sabouraud dextrose agar, subcultured in HiCrome™ agar, observed for the colonies of fungal elements, and were analyzed for species differentiation. RESULTS: There was a positive correlation between the presence of fungal elements and burning sensation in diabetic individuals, whereas it was not the case in the nondiabetic and prediabetic individuals. CONCLUSION: Candida colonization is one among a factor in diabetic individuals related with burning sensation of the mouth.

Clinicopathological Correlation of Pulp Stones and Its Association with Hypertension and Hyperlipidemia: An Hospital-based Prevalence Study.

INTRODUCTION: Pulp stones are the discrete calcification, located in pulp tissue or attached to or embedded in dentin. It occurs in physiological and pathological conditions. The exact etiopathogenesis of various types of pulp calcifications is unknown and the prevalence varies from 8% to 90%. The histopathological identification of pulp stones is higher than the radiological identification. OBJECTIVE: The aim of the study is to evaluate and correlate the clinical parameters and histopathological analysis of pulp stone with systemic hypertension and hyperlipidemia. MATERIALS AND METHODS: Seventy patients were selected for the study and a detailed case history was recorded. The radiological investigations were noted down and extirpated pulp tissues were sent for processing. The results were analyzed statistically using Chi-square test. RESULTS: Out of 70 patients studied, pulp stones were observed radiologically in 14 patients and histopathologically in 30 patients. The presence of irregular and nonlaminated type of pulp stones histopathologically was significantly correlated with hypertension and hyperlipidemia. CONCLUSION: The patients with the histopathological presence of nonlaminated and irregular-shaped pulp should be evaluated for lipid profile and hypertension.

Regenerative Capacity of Dental Pulp Stem Cells: A Systematic Review.

OBJECTIVES: The dental pulp contains undifferentiated mesenchymal cells, blood vessels and so on, which are responsible for routine functions of a tooth. The determination of stemness and regenerative properties using biomarkers and further application in routine practice may unravel its potential. MATERIALS AND METHODS: Inclusion criteria-original research articles published in English, from 2000 to 2019, were collected both manually and by electronic search from databases of Cochrane, Medline, Embase, and PubMed. Exclusion criteria-articles other than English and review manuscripts were omitted. The shortlisted articles were reviewed for specific biomarkers, to assess the regenerative potential, stemness, and lineage of dental pulp stem cells. RESULTS: Of 512 articles, 64 were selected and reviewed to determine the mesenchymal, neurogenic, vasculogenic, hematopoietic, and stem cell potential. On the basis of the search analysis, a panel of markers was proposed. CONCLUSION: The application of proposed markers, on a pulpectomized tissue derived from human teeth, may be helpful to determine the regenerative potential and the usefulness in regenerative medicine and tissue engineering.

COVID-19: Loss of bridging between innate and adaptive immunity?

COVID-19 has spread to most countries in the world. However, there are some striking differences in how COVID-19 is behaving in different age groups. While data on COVID-19 is limited, children appear to be less susceptible to severe disease. These unique characteristics may be considered as a potential link to understanding the immune system and response in COVID-19 and lead to an effective cure to the disease. We suggest a possible role of loss of bridging between innate and adaptive immunity in COVID-19 and a potential treatment modality also discussed.

Mesenchymal stem cells-bridge catalyst between innate and adaptive immunity in COVID 19.

Majority of patients infected with the COVID 19 virus display a mild to moderate course of disease and spontaneously recover at 14-20 days. However, about 15% of patients progress to severe stages and 2.5% of these patients succumb to this illness. Most patients with severe disease belong to the elderly age group (<65 years of age) and have multiple associated co-morbidities. The immune responses induced by the COVID 19 virus, during the incubation and non-severe stages, requires the early initiation of a specific adaptive immune response to eliminate the virus and prevent the progress to severe stages. In patients with a dysfunctional bridge adaptive immunity, the innate immune response becomes exaggerated due to the lack of feedback from the adaptive immune cells. The resultant cytokine storm is responsible for the severe lung injury leading to acute respiratory distress syndrome seen in COVID 19 patients. Mesenchymal stem cells are known to suppress overactive immune responses as well as bring about tissue regeneration and repair. This immuno-modulatory effect of MSCs could hold potential to manage a patient with severe symptoms of COVID 19 infection due to a dysfunctional adaptive immune system.

Correction: Sensory Neuron-Derived Eph Regulates Glomerular Arbors and Modulatory Function of a Central Serotonergic Neuron.

[This corrects the article DOI: 10.1371/journal.pgen.1003452.].

Sensory neuron-derived eph regulates glomerular arbors and modulatory function of a central serotonergic neuron.

Olfactory sensory neurons connect to the antennal lobe of the fly to create the primary units for processing odor cues, the glomeruli. Unique amongst antennal-lobe neurons is an identified wide-field serotonergic neuron, the contralaterally-projecting, serotonin-immunoreactive deutocerebral neuron (CSDn). The CSDn spreads its termini all over the contralateral antennal lobe, suggesting a diffuse neuromodulatory role. A closer examination, however, reveals a restricted pattern of the CSDn arborization in some glomeruli. We show that sensory neuron-derived Eph interacts with Ephrin in the CSDn, to regulate these arborizations. Behavioural analysis of animals with altered Eph-ephrin signaling and with consequent arborization defects suggests that neuromodulation requires local glomerular-specific patterning of the CSDn termini. Our results show the importance of developmental regulation of terminal arborization of even the diffuse modulatory neurons to allow them to route sensory-inputs according to the behavioural contexts.

Polymeric nanoparticles containing conjugated phospholipase A2 for nonviral gene delivery.

Polyethylenimine (PEI) was conjugated to phospholipase A(2) (PLA(2)) in an effort to improve transfection efficiency. PLA(2) was conjugated to PEI using EDC as a coupling reagent. The activity of enzyme in the conjugate was measured. DNA condensation ability of the conjugate to polymer was determined. The resultant nanoparticles were characterized by dynamic and electrophoretic light scattering. Two reporter genes were used to evaluate transfection efficiency in human embryonic kidney (HEK293) and human hepatoma (HepG2) cell lines. Conjugate was shown to retain PLA(2) activity and its ability to condense plasmid DNA, resulting in nanoparticles of a similar size to native PEI. The results demonstrated at N/P ratios of 15 and 20 showed 13- and 8-fold increase in transfection efficiency, respectively, compared to the maximum transfection efficiency of PEI (N/P ratio of 5) in the whole range of N/P ratios tested, from 5 to 60 in HepG2 cells. Toxicity studies in HepG2 cells showed uncomplexed conjugate had similar toxicity as PEI, and when complexed with DNA the conjugate had a significantly reduced toxicity. The results clearly indicate the potential for this approach to improve efficiencies of nonviral gene delivery vectors.

Evaluation of the transfection property of a peptide ligand for the fibroblast growth factor receptor as part of PEGylated polyethylenimine polyplex.

PURPOSE: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency. METHODS: The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using (1)H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay. RESULTS: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI. CONCLUSIONS: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.

Targeted polymers for gene delivery.

Over the past decade, significant research has been done in the area of polymer-mediated gene delivery. Synthesis of new polymers and modifications to existing polymers has resulted in polyplexes with improved in vitro and in vivo transfection efficiencies. Targeting has been an important aspect of this research, and various strategies for obtaining selective and enhanced gene delivery to the target site have been evaluated. This review covers the different aspects involved in polyplex targeting. Development of targeted polyplexes involves a careful consideration of the target site, the targeting ligand and the physicochemical properties of the polyplex itself. The need to redirect the polyplexes by using the 'shield and target' approach by reducing nonspecific interactions with negatively charged components, while conferring specificity by incorporating targeting ligands, is discussed. Basic chemistry involved in modifying polymers is covered and examples of targeting strategies used for tissue-specific gene delivery are discussed. Targeting is also discussed in the broader context of developing safe and effective polymeric vectors for in vivo gene delivery. Maximum benefit of targeting can be obtained when it is used as part of a multi-functional complex containing elements designed to improve gene delivery and reduce overall toxicity of the polyplex.