Investigating the chemo-preventive role of noscapine in lung carcinoma via therapeutic targeting of human aurora kinase B.

Lung carcinoma is the major contributor to global cancer incidence and one of the leading causes of cancer-related mortality worldwide. Irregularities in signal transduction events, genetic alterations, and mutated regulatory genes trigger cancer development and progression. Selective targeting of molecular modulators has substantially revolutionized cancer treatment strategies with improvised efficacy. The aurora kinase B (AURKB) is a critical component of the chromosomal passenger complex and is primarily involved in lung cancer pathogenesis. Since AURKB is an important therapeutic target, the design and development of its potential inhibitors are attractive strategies. In this study, noscapine was selected and validated as a possible inhibitor of AURKB using integrated computational, spectroscopic, and cell-based assays. Molecular docking analysis showed noscapine occupies the substrate-binding pocket of AURKB with strong binding affinity. Subsequently, MD simulation studies confirmed the formation of a stable AURKB-noscapine complex with non-significant alteration in various trajectories, including RMSD, RMSF, Rg, and SASA. These findings were further experimentally validated through fluorescence binding studies. In addition, dose-dependent noscapine treatment significantly attenuated recombinant AURKB activity with an IC50 value of 26.6 µM. Cell viability studies conducted on A549 cells and HEK293 cells revealed significant cytotoxic features of noscapine on A549 cells. Furthermore, Annexin-PI staining validated that noscapine triggered apoptosis in lung cancer cells, possibly via an intrinsic pathway. Our findings indicate that noscapine-based AURKB inhibition can be implicated as a potential therapeutic strategy in lung cancer treatment and can also provide a novel scaffold for developing next-generation AURKB-specific inhibitors.

Low monocytic HLA-DR expression in critically ill patients of sepsis: An indicator for antimicrobial and/or immunomodulatory intervention.

BACKGROUND: Sepsis is a result of suppressed host immune response which leads to fatal multi-organ dysfunctionality. Low frequency of active monocytes or reduced expression of human leukocyte antigen (HLA)-DR on monocytes shows the suppressed immune response in sepsis patients. One of the well-studied markers in patients with sepsis is procalcitonin (PCT). The role of monocytic (m) HLA-DR expression has been monitored in sepsis and is being considered a marker of the severity of interim immuno-depression in these patients. The study describes the impact of HLA-DR expression on monocytes quantitatively using flow cytometry. METHODS: In this prospective study, we quantified monocytes and their HLA-DR expression in 20 patients of sepsis admitted to the Intensive Care Unit (ICU). Serum levels of PCT and interleukin (IL)-6 production were also measured in these patients, and the results were compared with those in healthy controls. RESULTS: Monocyte frequency calculated was higher in sepsis patients as compared to healthy controls, however, HLA-DR expressing monocytes were significantly reduced as was the mean fluorescence intensity (MFI) of HLA-DR. Contrastingly, IL-6 and PCT levels were significantly high in sepsis than controls. The results suggest that low HLA-DR expression, combined with PCT, is a better prognostic parameter in the early phase of sepsis. CONCLUSION: Poor recovery of mHLA-DR may serve as an early guide for clinicians to assess the prognosis of sepsis patients and consider immunomodulatory therapy in its management.

Incompetent memory immune response in severe COVID-19 patients under treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated coronavirus disease 2019 (COVID-19) pandemic has affected millions of people worldwide and declared a Public Health Emergency by the World Health Organization (WHO) on January 30, 2020. Albeit, unprecedented efforts have been made from the scientific community to understand the pathophysiology of COVID-19 disease, the host immune and inflammatory responses are not explored well in the Indian population. Continuous arrival of new variants fascinated the scientists to understand the host immune processes and to eradicate this deadly virus. The aim of this study was to see the helper and cellular host immune responses including memory and activated cell subsets of COVID-19 patients admitted to the intensive care unit (ICU) at different time intervals during the treatment. PBMCs separated from nine patients with SARS-CoV-2 infection were incubated with fluorescent conjugated antibodies and acquired on flow cytometer machine to analyze the T and B cell subsets. The results in COVID-19 patients versus healthy volunteers were as follows: elevated helper T cells (57.4% vs 44.9%); low cytotoxic T cells (42.8% vs 55.6%), and activated T (17.7% vs 21.2%) subsets. Both, TREG (40.15% vs 51.7%) and TH17 (13.2% vs 24.6%) responses were substantially decreased and high expression of TREG markers was observed in these patients compared with controls.

Deregulated phenotype of autoreactive Th17 and Treg clone cells in pemphigus vulgaris after in-vitro treatment with desmoglein antigen (Dsg-3).

The loss of balance between regulatory T (Treg) and T helper 17 (Th17) causes loss of tolerance against desmoglein (Dsg)-3 leading to pemphigus vulgaris (PV), an autoimmune bullous skin disorder associated with autoantibodies against Dsg-3. We aimed to elucidate the complex relationship of Th17 and Treg cells, their molecules, and the underlying mechanism in the development of PV disease. Using cytokine secretion assays, Th17 and Treg cells were sorted by FACS Aria-III within Dsg-3-responsive PBMC population and homogeneous T cell clones were generated in-vitro. Different cell surface molecules like CD25, GITR, CD122, CD152, CD45RO, IL-23R, STAT3, STAT5, CD127, HLA-DR, CCR4, CCR5, CCR6 and CCR7 were studied. The functional response of Th17 and Treg cells were elucidated by measuring the levels of various cytokines released by IL-10 and IL-17 T cells. The mRNA expression of transcription factors (FoxP3 and RORγt) was also analyzed. IL-17 secreting (Th17) cells with phenotype CD4+IL-17+ were greatly increased and IL-10 secreting (Treg) cells with phenotype CD4+IL-10+ were reduced in PV cases than healthy controls. The qPCR analysis showing high expression of retinoic acid receptor-related orphan receptor gamma (RORγt) mRNA in comparison to forkhead box P3 (FoxP3) mRNA confirmed the development of pro-inflammatory Th17 response in PV. Further, the cytokine profile of pro-inflammatory and anti-inflammatory cytokines suggested defective suppressive functions in Treg cells with high inflammatory response. Our findings indicate that autoantigen Dsg-3 specifically allows the proliferation of IL-17 secreting T cells though has a negative effect on IL-10 secreting T cells leading to dysregulation of immunity in PV patients. This antagonistic relationship between Dsg-3-specific Th17 and Treg cells may be critical for the onset and persistence of inflammation in PV cases.

Implications of CD45RA and CD45RO T cell subsets in patients of chronic rhinosinusitis with nasal polyposis infected with Aspergillus flavus.

T cell subsets (CD4 and CD8) play a prominent role in the development of chronic rhinosinusitis with nasal polyposis (CRSwNP). Colonization with Aspergillus flavus is recognized as a trigger for the growth of nasal polyps. The fungal proteins initiate the recruitment of T cells into the nasal mucosa, which contributes to the progression of nasal polyps. The study included 50 cases of CRSwNP and 50 healthy controls. Biopsies were subjected to KOH and culture for mycological investigation. We examined the changes in T helper (CD4+) and T cytotoxic (CD8+) in total T cells (CD3+) and expression of naive (CD45RA) and memory (CD45RO) cell markers in T cell subsets in peripheral blood mononuclear cells (PBMCs) challenged by A. flavus antigens in cases before and after treatment and in healthy controls by flow cytometry. Predominantly, A. flavus (86%) identified in nasal polyp biopsies of patients. An increased percentage of CD3+CD4+ T cells observed after A. flavus stimulation in patients when compared with healthy controls. The expression of CD4+CD45RA+ cells was significantly (P < .05) reduced in patients and increased CD4+CD45RO+ was observed upon stimulation with A. flavus in patients when compared with healthy control. Continuous exposure to inhaled fungal spores may induce aberrant immune responses to A. flavus spores, causing an allergic immunological reaction with high CD4+T cell responses, resulting in an unfavourable outcome. Elevated CD4+CD45RO+ T cells may transform the pathogenic response and highlight the chances of A. flavus reactive T cells involvement in prompting inflammation in CRSwNP.

TLR-2 expression and dysregulated human Treg/Th17 phenotype in Aspergillus flavus infected patients of chronic rhinosinusitis with nasal polyposis.

BACKGROUND: T helper (Th)17 and regulatory T (Treg) cells with toll-like receptor (TLR)-2 have been acknowledged to play a critical role in chronic rhinosinusitis with nasal polyposis (CRSwNP). However, its pathogenesis has been perplexed by conflicting reports on the role of Th17/Treg cells in patients of distinct ethnicities. We attempted to understand the role of Th responses induced during host defense against Aspergillus flavus. RESULTS: The percentages of Th17 (CD4+CD161+IL23R+) and Treg (CD4+CD25+FoxP3+) cell populations and various cytokine profiles in peripheral blood mononuclear cells (PBMCs) challenged by A. flavus antigens were characterized from 50 CRSwNP cases, before and after treatment, and in 50 healthy controls. TLR-2 expression was analyzed in tissues of cases and controls for disease co-relation. The major pathogen identified in our study was A. flavus by mycological investigations. A marked immune imbalance was noted with elevated Th17 and decreased Tregs in PBMCs of CRSwNP patients after A. flavus stimulation. Comparatively, interleukin (IL)-17 and IL-10 levels were increased, with low transforming growth factor (TGF)-ß levels in A. flavus stimulated PBMC supernatants of patients. The mRNA expression of TLR-2 in polyps of CRSwNP patients indicated significant (p = 0.001) upregulation in comparison to the controls. CONCLUSIONS: Our data highlights the excessive expression of TLR-2 in nasal polyps contributing to the imbalance in Th17/Tregs population in patients. After therapy, recovery of Tregs cells indicates restoration and tissue homeostasis, though high circulating CD4+CD161+ Th17 cells may continue to be a threat to patients predisposed to future recurrences. The constant exposure and tendency of A. flavus to colonize nasal cavities can lead to a Th17 driven airway inflammation. Dysregulated Th17 with TLR-2 promote resistance to treatment and progression to the chronicity of the disease.

Digital Assessment of Difficulty in Impacted Mandibular Third Molar Extraction.

PURPOSE: In recent era of computer and software technology, it is necessary to introduce software which helps in routine assessment of surgical procedures practiced in oral surgery. Removal of impacted third molar is a common procedure. It is hard to evaluate factors that complicate removal of impacted third molars because of the large variation among patients and the difficulty in creating a study design. In this article, we have described about our newly designed software developed in order to assess the difficulty in extracting impacted mandibular third molars accurately, thereby reducing the bias faced during the assessment of difficulty in removing impacted mandibular third molar. MATERIALS AND METHOD: A software is designed using C# computer language and Windows Presentation Foundation Framework. RESULTS: The measurements and angulations are accurately calculated by this software which helps to bring about uniformity in results, thus minimizing the bias during clinical as well as study purposes. CONCLUSION: Mandibular third molar difficulty level calculator can be useful software for dental practitioners in day-to-day practice. Dental students and professionals should be made aware of this software so as to utilize it to the utmost possible level.

The interplay among Th17 and T regulatory cells in the immune dysregulation of chronic dermatophytic infection.

The delineation of the pathogenic interaction between the host skin immune responses and dermatophytes has remained indigent. The obscure enigma in host-dermatophyte immunopathogenic interactions is the T regulatory (Treg) and T-helper (Th) 17  cell role in maintaining immune homeostasis. We attempted to understand the regulation and recognition of lineage-specific response in chronic dermatophytic skin infection patients. The percentages of Th17 (CD4+CD161+IL23R+) and Treg (CD4+CD25+FoxP3+) cell subpopulations in the peripheral circulation of thirty chronic dermatophytic skin infection patients and twenty healthy individuals was determined. The serum cytokine levels were estimated for disease correlation. The mean duration of the disease was 10.68 ± 8.72 months, with Trichophyton mentagrophytes complex as the major pathogen. Total serum IgE level of patients was significantly higher compared to healthy controls (305 ± 117 vs 98.53 ± 54.55 IU/ml; p < 0.01). Expression of Th17 and Treg cell markers on CD4+ T cells was significantly elevated in patients than controls (p < 0.05). Comparatively, serum interleukin (IL)-4 and interferon (IFN)-γ levels were increased, with low IL-10 levels in patients. Our data envisages a complex immune dysfunction in chronic dermatophytosis, arising either as a result of dermatophyte exposure or paradoxical precedence of disease establishment. Designing new treatment strategies and preventing recurrences are challenges for future research.

Phenotypic and functional profile of Th17 and Treg cells in allergic fungal sinusitis.

Interleukin-17 producing T helper (Th17) and regulatory T cells (Treg) cells have been identified to play a critical role in atopic inflammation. However, conflicting reports on the role of Th17/Treg cells in allergic fungal rhinosinusitis (AFRS) patients of different ethnicities has mystified its pathogenesis. To better understand the pathophysiological mechanisms involved in AFRS, we conducted a prospective, analytical, case-control study involving 40 confirmed immunocompetent AFRS patients and 20 healthy controls. The distribution of Th17 and Treg cells in PBMC, intracellular mRNA expression of retinoid orphan nuclear receptor (RORγt) in Th17 and forkhead transcription factor (FoxP3) in Treg cells, and serum cytokine levels were investigated. Aspergillus flavus was identified from majority (85%) of patient tissue biopsies. Total serum IgE level along with cytokines IL-17, IL-21, IL-1ß and TGF-ß were comparatively elevated in AFRS. Nevertheless, IL-2 and IL-10 were reduced. Higher percentages of CD3+CD4+ T cells in AFRS with increased expression of CD161 and/or IL-23R markers were observed. Though, lower percentages of CD4+CD25+ Treg cells with elevated expression of GITR were patent. Transcription factor RORγt mRNA was upregulated, whereas FoxP3 mRNA was downregulated in AFRS patients. This inclination of Th17/Treg balance towards Th17, and the proposed role of Tregs on Th1 and Th2 cells in AFRS, directed us to conclude that Aspergillus infestation may lead to development of atopy and immunological dysbalance inciting a Th17 driven response, thereby, promoting aggravation of nasal polyposis. The observation may provide new insight into the molecular mechanisms leading to revision of the classical paradigm.

Serum Cytokine Profile in Patients with Chronic Rhinosinusitis with Nasal Polyposis Infected by Aspergillus flavus.

BACKGROUND: Fungi, especially Aspergillus flavus, can cause chronic rhinosinusitis with nasal polyposis and modulate host innate immune components. The objective of this study was to examine the serum levels of T helper (Th) cell subset Th1, Th2, and Th17 cytokines and total IgE in patients having chronic rhinosinusitis with nasal polyposis and Aspergillus flavus infection. METHODS: A case-control study including 40 patients with chronic rhinosinusitis with nasal polyposis and 20 healthy controls was conducted. Aspergillus flavus infection was confirmed by standard potassium hydroxide (KOH) testing, culture, and PCR. Serum samples of all patients and controls were analyzed for various cytokines (interleukins [IL]-1ß, IL-2, IL-4, IL-6, IL-17, IL-21, IL-27, TGF-ß) and total IgE by ELISA. Data from patients with Aspergillus flavus infection and healthy volunteers were compared using the independent t-test and non-parametric Mann-Whitney U test. RESULTS: Aspergillus flavus infection was found in 31 (77.5%) patients with chronic rhinosinusitis with nasal polyposis. IL-1ß, IL-17, IL-21, and TGF-ß serum levels were significantly higher in these patients than in controls; however, IL-2, IL-4, IL-6, and IL-27 levels were lower. Compared with nine (22.5%) patients without Aspergillus flavus infection, IL-17 level was higher while IL-2 level was lower in patients with Aspergillus flavus infection. Total IgE was significantly higher in patients with Aspergillus flavus infection than in controls. CONCLUSIONS: High levels of IL-17 and its regulatory cytokines in patients with chronic rhinosinusitis with nasal polyposis infected by Aspergillus flavus raise a concern about effective disease management and therapeutic recovery. Surgical removal of the nasal polyp being the chief management option, the choice of post-operative drugs may differ in eosinophilic vs. non-eosinophilic nasal polyposis. The prognosis is likely poor, warranting extended care.

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review.

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.

Novel single-step multiplex real-time polymerase chain reaction assay for simultaneous quantification of hepatitis virus A, B, C, and E in serum.

BACKGROUND AND AIM: Viral hepatitis needs an earliest diagnosis for its proper and timely treatment. Although serodiagnosis of viral hepatitis is in regular practice, however, it has certain limitations and points to alternate procedures of diagnosis. Present study was designed to develop a single-step multiplex real-time polymerase chain reaction (PCR) assay for detection of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus (HEV) related nucleic acids in sera from infected patients. METHODS: The PCR was standardized to detect HAV, HBV, HCV and HEV in serum using variables including annealing temperature, extension temperature, MgCl2 , and primer concentrations. The conserved regions of all viral genomes were used as targets for amplification. RESULTS: This novel assay was found to be a fast, sensitive, specific, and reproducible system for detection of HAV, HBV, HCV, and HEV in serum. The detection limit for different viral genomes at 100% level was found to be 280 copies/mL for HAV, 290 copies/mL for HBV, 30 copies/mL for HCV, and 300 copies/mL for HEV in a single-tube assay system. CONCLUSION: Present multiplex real-time PCR is the first report on single-step nucleic acid detection of HAV, HBV, HCV, and HEV in sera samples. It is an alternate diagnostic assay for common use in laboratories analyzing viral hepatitis cases.