Extraction and characterization of exosomes from the exhaled breath condensate and sputum of lung cancer patients and vulnerable tobacco consumers-potential noninvasive diagnostic biomarker source.

Noninvasive sample sources of exosomes, such as exhaled breath and sputum, which are in close proximity to the tumor microenvironment and may contain biomarkers indicative of lung cancer, are far more permissive than invasive sample sources for biomarker screening. Standardized exosome extraction and characterization approaches for low-volume noninvasive samples are critically needed. We isolated and characterized exhaled breath condensate (EBC) and sputum exosomes from healthy nonsmokers (n= 30), tobacco smokers (n= 30), and lung cancer patients (n= 40) and correlated the findings with invasive sample sources. EBC samples were collected by using commercially available R-Tubes. To collect sputum samples the participants were directed to take deep breaths, hold their breath, and cough in a collection container. Dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy were used to evaluate the exosome morphology. Protein isolation, western blotting, exosome quantification via EXOCET, and Fourier transform infrared spectroscopy were performed for molecular characterization. Exosomes were successfully isolated from EBC and sputum samples, and their yields were adequate and sufficiently pure for subsequent downstream processing and characterization. The exosomes were confirmed based on their size, shape, and surface marker expression. Remarkably, cancer exosomes were the largest in size not only in the plasma subgroups, but also in the EBC (p < 0.05) and sputum (p= 0.0036) subgroups, according to our findings. A significant difference in exosome concentrations were observed between the control sub-groups (p < 0.05). Our research confirmed that exosomes can be extracted from noninvasive sources, such as EBC and sputum, to investigate lung cancer diagnostic biomarkers for research, clinical, and early detection in smokers.

Enhancing Proliferation of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) through hTERT Expression while Preserving Stemness and Multipotency.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) hold promise in regenerative medicine owing to their multipotent capabilities resembling mesenchymal stem cells (MSCs). Despite their potential, SHED have not been extensively investigated because their limited lifespan and unavailability of cell-lines pose challenges for therapeutic applications. This study investigated the effect of ectopic human telomerase reverse transcriptase (hTERT) expression on SHEDs' proliferation while preserving stemness and genomic integrity. METHODS: Deciduous teeth were collected from children aged 6-10 years. After isolation and characterization, the SHED were transduced with pBabe-puro-hTERT retrovirus to establish SHED cell-line, which was evaluated and compared with pBabe-puro (mock control) for stemness, multipotency and growth attributes through flow cytometry, trilineage differentiation, and growth kinetics. We also estimated hTERT gene expression, genomic integrity, and validated cell-line through STR analysis. RESULTS: Following hTERT transduction, SHED displayed elevated hTERT gene expression while retaining fibroblast-like morphology and mesenchymal stem cell markers. Moreover, after hTERT transduction cellular shape remained same along with increased replicative lifespan and proliferation potential. SHED-hTERT cells exhibited multi-potency and maintained stemness, as evidenced by surface marker expression and multilineage differentiation. Furthermore, genomic integrity was not affected by hTERT integration, as confirmed by STR analysis and CDKN2A gene assessment. CONCLUSION: Ectopic hTERT expression in SHED successfully prolonged their replicative lifespan and improved their ability to proliferate and migrate, while preserving their stemness, multipotency and genomic integrity, suggesting minimal carcinogenic risk. Establishment of SHED cell-line holds potential in regenerative medicine applications, especially in cell-based drugs and tissue engineering experiments.

Exploring salivary exosomes as early predictors of oral cancer in susceptible tobacco consumers: noninvasive diagnostic and prognostic applications.

BACKGROUND: Salivary exosome analysis provides a noninvasive and comprehensive approach with potential applications in oral cancer diagnosis and prognosis. The early detection of oral cancer has remained a critical concern in enhancing the quality of life, especially for individuals who consume tobacco and are at greater risk of developing the disease. The current study investigates the potential of salivary exosomes in screening smokers for early signs and transformations of oral cancer. METHODS: Morphological characterization of salivary exosomes among three study groups (non-smokers as control, smokers as high-risk tobacco consumers, and Oral cancer) (n = 120) was carried out through dynamic light scattering, and nanoparticle tracking analysis. For molecular characterization, EXOCET and Fourier transform infrared spectroscopy methods were utilized. The expression of the exosomal surface protein CD63 was evaluated using Western blotting. RESULTS: Salivary exosomes exhibit noticeable differences in size between control group and tobacco consumers. The differentiation extended beyond exosome size and included variations in concentration and bio-molecular composition, as determined by FTIR screening. Tobacco consumers and oral cancer groups showed significantly larger and more concentrated exosomes compared to the healthy group. CONCLUSION: Our study provides strong evidence that the properties of salivary exosomes can serve as reliable noninvasive biomarkers for distinguishing tobacco consumers from non-smokers and oral cancer patients. Our results underscore the potential of exosome-based diagnostics in early oral cancer detection for high-risk individuals. The larger size and higher concentration of exosomes in tobacco consumers indicate early changes in cell secretions associated with the transformation from healthy to abnormal cells.

Cyclodextrin-Based Arsenal for Anti-Cancer Treatments.

Anti-cancer drugs are mostly limited in their use due to poor physicochemical and biopharmaceutical properties. Their lower solubility is the most common hurdle limiting their use upto their potential. In the recent years, the cyclodextrin (CD) complexation have emerged as existing approach to overcome the problem of poor solubility. CD-based nano-technological approaches are safe, stable and showed well in vivo tolerance and greater payload for encapsulation of hydrophobic drugs for the targeted delivery. They are generally chosen due to their ability to get self-assembled to form liposomes, nanoparticles, micelles and nano-sponges etc. This review paper describes a birds-eye view of the various CD-based nano-technological approaches applied for the delivery of anti-cancer moieties to the desired target such as CD based liposomes, niosomes, niosoponges, micelles, nanoparticles, monoclonal antibody, magnetic nanoparticles, small interfering RNA, nanorods, miscellaneous formulation of anti-cancer drugs containing CD. Moreover, the author also summarizes the various shortcomings of such a system and their way ahead.

Exosomics in oral cancer diagnosis, prognosis, and therapeutics - An emergent and imperative non-invasive natural nanoparticle-based approach.

Exosomes- the natural nanoparticles belonging to heterogeneous vesicles are released via nearly all sorts of cells, including tumour cells, to oprate intercellular communication. Selective packaging of exosomes amid nucleic acids, phospholipids, and proteins makes them ideal for intercellular communications occurring among different cells. The existence of exosomes has been validated in various biofluids, including saliva. Being non-invasive and in direct contact with oral malignant cells, saliva establishes itself as a preeminent source of early cancer biomarkers. In context, the role and providence of both recipient and donor secreting cells are persuaded through exosomal cargo.Several studies have emphasized the influence of exosomal contents in different stages of cancer development, reconciling interactions between tumour cells and their surrounding niche. More explicitly, a transformation of exosomal contents such as nucleic acids, lipids, and proteins can endorse tumour progression and help ascertain a secluded pre-metastatic niche crammed with substances that errand cancer cell proliferation,angiogenesis, metastasis, and drug resistance. The blooming field of exosomes has directed the evolution of high-end isolation and characterization techniques along with the development of an entirely new field- exosomics that comprises complete analysis of exosomal cargo in various physiological conditions, including oral cancer. Researchers have discovered multiple pathways involved in exosome biogenesis to understand numerous events associated with cancer progression. Tissue-specific packaging of exosomes makes them a novel source of prognostic and diagnostic biomarkers and potential therapeutic targets. The extent of the current review confers the contemporary perception of the versatile task of exosomes, especially salivary exosomes, as potential biomarkers in the progression and diagnosis as well as therapeutics of oral cancers and their potential employment in clinical applications.

Effect of fetal liver condition media derived cytokines(IL-6 and Flt-3) on human bone marrow stem cells colony formation.

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA). In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.

A Phytochemicals Approach Towards the Treatment of Cervical Cancer Using Polyphenols and Flavonoids.

AIM: Despite enormous progress in cancer biology, oncologists are still struggling to retrieve the methods and drugs to cure cancer which remains a global threat to humans. Plant-derived natural compounds, also known as phytochemicals, carry therapeutic potential and could be taken as dietary supplements, which are a radical way to resist as well as cure cancer. The present study reveals the anti-cancerous potential of a few phytochemical constituents under in vitro conditions and their mode of action on cervical cancer. SiHa was treated with phytochemicals viz. Quercetin dihydrate, Gallic Acid, and Naringin with varying concentrations to assess their cytotoxicity potential by various methods and also to elucidate their IC50 values. METHODS: All three compounds reduced the cell number and viability, along with alterations in cancer cell morphology. The diagnosed IC50 values of the compounds were 160µM, 200µM, and 1500µM for Quercetin dihydrate, Gallic Acid, and Naringin, respectively. DNA fragmentation assay and gene expression analysis were also used to assess apoptosis and anti-proliferative activity of compounds. RESULTS: We found fragmented DNA in treated cells as assessed by gel electrophoresis assay. These phytochemicals elicit an apoptotic response in SiHa cells by significantly up-regulating the gene expression level of p53 and p21 (p-value <0.005). CONCLUSION: Considering the anti-cancer and anti-proliferative potential of Quercetin dihydrate, Gallic Acid, and Naringin on the cervical cancer cell line, these phytochemicals could be used as an alternative or concurrent cancer therapeutic approach. However, further in-depth elucidation of their mode of action, safety, and efficacy should be explored.

Cytotoxic Role of Chlorogenic Acid on Oral Squamous Cell Carcinoma Cell Line.

The aim of the present study was to determine the cytotoxic, anticancerous and antiproliferative activity of CGA on oral squamous cell carcinoma (OSCC) cell line (KB) and to evaluate expression level of p21 and p53 in these CGA treated OSCC cell line. Different concentrations of CGA varying from 500 to 2500 µM were tested on OSCC cell line. Trypan blue and MTT assay were performed to establish IC50. DNA fragmentation and expression level of p21 and p53 were evaluated with the help of RT-PCR. CGA exerted antiproliferative and cytotoxic effect on OSCC (KB) cell line. Statistically significant results were found regarding effect of different CGA concentrations on KB cell line with IC50 at 1800 µM. No DNA fragmentation was observed. p21 and p53 expression were down regulated after CGA treatment. CGA revealed neither apoptosis nor damage to the nucleus after DNA fragmentation. Antiproliferative role of CGA was hinted by down regulation of p53 and p21 probably through cell cycle arrest at G1-S phase. It was reaffirmed that CGA a natural chemo preventive agent could enhance the treatment modalities with minimal side effects.

Application of the Bethesda system of reporting for cervical cytology to evaluate human papilloma virus induced changes in oral leukoplakia, oral squamous cell carcinoma, and oropharyngeal squamous cell carcinoma: A cytomorphological and genetic study.

BACKGROUND: Human papilloma virus (HPV) has a well-established carcinogenic role in certain head and neck cancers. These HPV associated cancers possess unique clinicopathological behavior and exhibits better prognosis than their negative counterparts. Detection through polymerase chain reaction (PCR) has been considered as the "gold standard" but imposes burden in low resource settings. Therefore, in the present study, we assessed the validity of cytomorphological features for the detection of HPV in oral leukoplakia (OL), oral squamous cell carcinoma (OSCC), and oropharyngeal squamous cell carcinoma (OPSCC). METHODOLOGY: This study included 63 subjects comprising of 25 OL, 26 OSCC, and 12 OPSCC cases. Exfoliated cells were collected and processed for PCR followed by Papanicolaou staining and subsequent grading. Additionally the non-classical signs were evaluated and statistical analysis included Chi-square and Spearman's test. RESULT: 23/63 (36.5%) cases showed PCR positivity for HPV16. Most of the cytomorphological features showed significant correlation for the presence of HPV. A greater sensitivity and specificity was observed in the Bethesda system for reporting cervical cytology (TBS) than the Papanicolaou grading system. CONCLUSION: We conclude that the non-classic cytological features could be employed in the detection of HPV in low resource settings with improved sensitivity. Liquid based cytology graded using TBS could be suitable for oral cytology in the detection of early atypical changes.

Bioactive compounds and their libraries: An insight into prospective phytotherapeutics approach for oral mucocutaneous cancers.

Oral mucocutaneous cancers (OMCs) are cancers that affect both the oral mucosa and perioral cutaneous structures. Common OMCs are squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and malignant melanoma (MM). Anatomical similarities and conventions which categorizes these lesions blur the magnitude of OMCs in diverse populations. The burden of OMC is high in the sub-Saharan Africa and Indian subcontinents, and the cost of management is prohibitive in the resource-limited, developing world. Hence, there is a pressing demand for the use of cost-effective in silico approaches to identify diagnostic tools and treatment targets for diseases with high burdens in these regions. Due to their ubiquitousness and accessibility, the use of therapeutic efficacy of plant bioactive compounds in the management of OMC is both appropriate and plausible. Furthermore, screening known mechanistic disease targets with well annotated plant bioactive compound libraries is poised to improve the routine management of OMCs provided that the requisite access to database resources are available and accessible. Using natural products minimizes the side effects and morbidities associated with conventional therapies. The development of innovative treatments approaches would tremendously benefit the African and Indian populace and reduce the mortalities associated with OMCs in the developing world. Hence, we discuss herein, the potential benefits, opportunities and challenges of using bioactive compound libraries in the management of OMCs.

In vitro expansion of fetal liver hematopoietic stem cells.

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

Molecular and Immunohistochemical Cognizance of HPV16 in Oral Leukoplakia, Oral Squamous Cell Carcinoma and Oropharyngeal Squamous Cell Carcinoma.

Prior studies have established the carcinogenic role of HPV16 and also demonstrated its unique biological behavior in cervical and oropharyngeal squamous cell carcinoma (OPSCC) but its role in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) is not well explored. Therefore, in the present study, we assessed HPV16 prevalence using PCR and Anti-HPV16 antibodies for the first time and correlated its biological behavior using p16INK4a and Ki67 proliferation index (PI) in OL, OSCC, and OPSCC. This study included 63 subjects comprising of 25 OL, 26 OSCC, and 12 OPSCC cases. Exfoliated cells were collected and processed for PCR followed by immunohistochemistry with primary antibodies p16INK4a, Anti-HPV16, and Ki67. The expressions were evaluated and statistical analysis included Chi-square and Spearman's test. Cumulatively 37% (OL-7%, OSCC-14% & OPSCC-16%) of cases showed positive PCR expression. PCR positivity was observed to be significantly higher (p 0.00) in OPSCC (9/12) than OSCC (9/26) and OL (5/25) cases. Overall immunohistochemical expression of p16INK4a, Anti-HPV16, and Ki67 were significantly (p 0.02) higher in HPV16 (PCR) positive cases. HPV16 + OSCC cases showed higher grades of p16INK4a and Ki67 expression. We have demonstrated a prevalence of HPV16 in OL, OSCC, and OPSCC through PCR, which may be concluded as a gold standard for the detection of HPV16 DNA.

Mesenchymal stem cell immunomodulation and regeneration therapeutics as an ameliorative approach for COVID-19 pandemics.

The severe acute respiratory syndrome-novel coronavirus mediated COVID-19 has been recently declared a pandemic by the World Health Organization. The primary target of the SARS-CoV-2 virus is the human lungs governed by the ACE-2 receptor of epithelial type II cells/endothelial cells, which promote modulation of the immune response of host cells through generating cytokine storm, inflammation, severe pneumonia symptoms, and secondary complications such as acute respiratory distress syndrome. Although numerous antiviral and anti-parasitic drugs are under clinical trials to combat this pandemic, to date, neither a specific treatment nor any successful vaccine has been established, urging researchers to identify any potential candidate for combating the disease. Mesenchymal stem cells own self-renewal, differentiation, homing, immunomodulation and remains unaffected by the coronavirus on the virtue of the absence of ACE-2 receptors, indicating that MSC's could be used an ameliorative approach for COVID-19. MSCs have shown to combat the disease via various pathways such as repairing the lung epithelial and endothelial cells, reducing hyperimmune response, maintaining the renin-angiotensin system. Although MSCs-based treatment approaches for COVID-19 is still under consideration with limited data, many human clinical trials of MSC's has been initiated to explore their potential for COVID 19 treatment. The current review summarizes and emphasizes on how MSC's modulate the immune response, can repair the lungs from the impact of the virus, and various aspects of MSC's as a remedial source for COVID-19, to provide better insight for biomedical researchers and for those who are fascinated by stem cells as a therapeutic approach.

Hematopoietic Stem Cells Culture, Expansion and Differentiation: An Insight into Variable and Available Media.

Owing to differentiation and self-renewal capacity, hematopoietic stem cells clasp potentiality to engender all blood cell types, leading to their immense competence to play a diverse role in therapeutic applications. Although these stem cells are the most investigated and exploited until now, further research is still essential to comprehend their nature, fate, and potential. Enhanced usage of hematopoietic stem cells in research and therapeutics intensified the requirement of expansion and differentiation of hematopoietic stem cells under in vitro conditions. Since these cells remain in senescence for a prolonged period before isolation, selection of appropriate growth medium along with supplements and culture conditions are crucial to initiate their cell division and to designate their destiny. The precise equilibrium between self-renewal and differentiation of stem cells sustained by exclusive medium along with special growth or differentiation factors is accountable for generating diverse cell lineages. Maintenance of hematopoietic stem and progenitor cell lines along with the advancement of research work generate an inexorable demand for production and commercialization of specialized stem cell culture media, with or without serum along with specific growth factors and supplements. Media commercialization for precise stem cell types, culturing and differentiation is a cost-effective developing field. Here in this review, we are assembling various types of hematopoietic stem cell self-renewal, expansion and differentiation media along with supplements and culture conditions, either developed and used by various scientists or are available commercially.

Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice.

The myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

Patients' perspectives on timing of urinary catheter removal after surgery.

UNLABELLED: A prolonged catheter duration is a major risk factor for catheter-associated urinary tract infection, with bacteriuria increasing by 5% per day (Gokula et al, 2004). AIM: In this study, the authors explored patients' perceptions of the care process relating to peri-operative catheterisation to identify patient factors that encourage early removal. METHOD: Semi-structured interviews, incorporating a grounded theory approach, were performed on three men and seven women during 2010. Interviews were transcribed and analysed using constant comparative method and thematic framework analysis. RESULTS: Catheter duration ranged 1-10 days. Main themes elicited included: lack of understanding of the purpose and catheterisation process; loss of patient autonomy and dignity; and impact of environmental factors. CONCLUSION: Lack of knowledge of the catheterisation process among participants led to fears and concerns that may have contributed to delayed catheter removal. Changes to patient care that are likely to reduce catheter duration include ensuring the provision of pre-operative information, greater patient involvement in catheter removal decisions, and provision of easily accessible toilet facilities.

Reduced ribosomal protein gene dosage and p53 activation in low-risk myelodysplastic syndrome.

Reduced gene dosage of ribosomal protein subunits has been implicated in 5q- myelodysplastic syndrome and Diamond Blackfan anemia, but the cellular and pathophysiologic defects associated with these conditions are enigmatic. Using conditional inactivation of the ribosomal protein S6 gene in laboratory mice, we found that reduced ribosomal protein gene dosage recapitulates cardinal features of the 5q- syndrome, including macrocytic anemia, erythroid hypoplasia, and megakaryocytic dysplasia with thrombocytosis, and that p53 plays a critical role in manifestation of these phenotypes. The blood cell abnormalities are accompanied by a reduction in the number of HSCs, a specific defect in late erythrocyte development, and suggest a disease-specific ontogenetic pathway for megakaryocyte development. Further studies of highly purified HSCs from healthy patients and from those with myelodysplastic syndrome link reduced expression of ribosomal protein genes to decreased RBC maturation and suggest an underlying and common pathophysiologic pathway for additional subtypes of myelodysplastic syndrome.