The epidemiology of superficial Streptococcal A (impetigo and pharyngitis) infections in Australia: A systematic review.

BACKGROUND: Streptoccocal A (Strep A, GAS) infections in Australia are responsible for significant morbidity and mortality through both invasive (iGAS) and post-streptococcal (postGAS) diseases as well as preceding superficial (sGAS) skin and throat infection. The burden of iGAS and postGAS are addressed in some jurisdictions by mandatory notification systems; in contrast, the burden of preceding sGAS has no reporting structure, and is less well defined. This review provides valuable, contemporaneous evidence on the epidemiology of sGAS presentations in Australia, informing preventative health projects such as a Streptococcal A vaccine and standardisation of primary care notification. METHODS AND FINDINGS: MEDLINE, Scopus, EMBASE, Web of Science, Global Health, Cochrane, CINAHL databases and the grey literature were searched for studies from an Australian setting relating to the epidemiology of sGAS infections between 1970 and 2020 inclusive. Extracted data were pooled for relevant population and subgroup analysis. From 5157 titles in the databases combined with 186 grey literature reports and following removal of duplicates, 4889 articles underwent preliminary title screening. The abstract of 519 articles were reviewed with 162 articles identified for full text review, and 38 articles identified for inclusion. The majority of data was collected for impetigo in Aboriginal and Torres Strait Islander populations, remote communities, and in the Northern Territory, Australia. A paucity of data was noted for Aboriginal and Torres Strait Islander people living in urban centres or with pharyngitis. Prevalence estimates have not significantly changed over time. Community estimates of impetigo point prevalence ranged from 5.5-66.1%, with a pooled prevalence of 27.9% [95% CI: 20.0-36.5%]. All studies excepting one included >80% Aboriginal and Torres Strait Islander people and all excepting two were in remote or very remote settings. Observed prevalence of impetigo as diagnosed in healthcare encounters was lower, with a pooled estimate of 10.6% [95% CI: 3.1-21.8%], and a range of 0.1-50.0%. Community prevalence estimates for pharyngitis ranged from 0.2-39.4%, with a pooled estimate of 12.5% [95% CI: 3.5-25.9%], higher than the prevalence of pharyngitis in healthcare encounters; ranging from 1.0-5.0%, and a pooled estimate of 2.0% [95% CI: 1.3-2.8%]. The review was limited by heterogeneity in study design and lack of comparator studies for some populations. CONCLUSIONS: Superficial Streptococcal A infections contribute to an inequitable burden of disease in Australia and persists despite public health interventions. The burden in community studies is generally higher than in health-services settings, suggesting under-recognition, possible normalisation and missed opportunities for treatment to prevent postGAS. The available, reported epidemiology is heterogeneous. Standardised nation-wide notification for sGAS disease surveillance must be considered in combination with the development of a Communicable Diseases Network of Australia (CDNA) Series of National Guideline (SoNG), to accurately define and address disease burden across populations in Australia. TRIAL REGISTRATION: This review is registered with PROSPERO. Registration number: CRD42019140440.

Changes in quality of life over time in patients diagnosed with oral leukoplakia: A prospective longitudinal study.

BACKGROUND: Oral leukoplakia (OL) is one of the most prevalent oral potentially malignant disorders (OPMD). Although there is emerging evidence that quality of life (QoL) is impaired in subjects with OL; studies to date are based on single and heterogenous point-in-time assessments. The aim of this study was to ascertain if QoL scores change over time in individuals diagnosed with OL. METHODS: Forty-one individuals with OL were administered the Short Generic Health Questionnaire (SF-12) and the discipline-specific Oral Potentially Malignant Disorder Questionnaire (OPMDQ) at four points in time: at the time of clinical diagnosis, at the post-biopsy review (confirmed diagnosis), and at 3- and 6-month follow-up appointments. Responses were compared between the four time points. RESULTS: No significant differences were observed in the SF-12 questionnaire scores over time. However, a general improvement in the participants' life quality was evident over the 6-month period under investigation in the domains of psychological and social well-being (p = 0.0027) and effect of treatment on daily life (p = 0.0317) as well as in the total score (p = 0.0005) of the OPMDQ. Age, gender, medical status, tobacco/alcohol use, lesion site, size, the presence of dysplasia and treatment did not impact QoL scores over time. CONCLUSIONS: QoL scores of OL subjects may improve with time. Our results suggest that studies examining QoL in individuals with OL should be controlled for time at which the participants are surveyed.

Breath biopsy, a novel technology to identify head and neck squamous cell carcinoma: A systematic review.

Head and neck cancers are a heterogeneous group of neoplasms, which together comprise the sixth most common cancer globally. Breath biopsies are a non-invasive clinical investigation that detect volatile organic compounds (VOCs) in exhaled breath. This systematic review examines current applications of breath biopsy for the diagnosis of head and neck squamous cell carcinoma (HNSCC), including data on efficacy and utility, and speculates on the future uses of this non-invasive detection method. Medline, PubMed, Web of Science, Cochrane and Scopus, as well as the grey literature were searched using a search strategy developed to identify relevant studies on the role of breath biopsy in the diagnosis of HNSCC. All included studies were subject to a thorough methodological quality assessment. The initial search generated a total of 1443 articles, 20 of which were eligible for review. A total of 660 HNSCC samples were investigated across the included studies. 3,7-dimethylundecane and benzaldehyde were among several VOCs to be significantly correlated with the presence of HNSCC compared to healthy controls. We show that current breath biopsy methods have high accuracy, specificity and sensitivity for identifying HNSCC. However, further studies are needed given the reported poor quality of the included studies.

The role of Twitter in dental education: A systematic review.

BACKGROUND: The rapid growth of social media in recent years has highlighted uses beyond their original purposes, particularly in education. Twitter is a free, open access social network with high potential to enhance interactive learning. The use of Twitter in dental education has been far less investigated; therefore, the objective of this systematic review is to explore the current uses and to examine the impact of Twitter on dental education, and to analyze and predict potential models of Twitter for future application in dental training, education, and teaching. METHODS: Five databases (PubMed, Embase, Medline, Scopus, and Web of Science) and the gray literature using keywords related to Twitter and dental education were searched. Articles were screened for inclusion, and two researchers independently extracted the data using a standardized data collection template and analyzed the quality of the included articles using the Medical Education Research Study Quality Instrument. RESULTS: Of the 121 articles identified from the initial search, 68 remained after duplications were removed. Article screening removed 61 articles leaving 7 eligible for inclusion and data extraction. Five studies were cross-sectional and two were cohort studies, and all involved survey-based designs with 998 respondents in total. Quality assessment gave a score range between 8 and 12.5 out of a total of 18 points. CONCLUSIONS: Our study supports the potential for Twitter as a useful learning tool in dental education. Features, including the open access nature of Twitter as well as the low level of ads and free registration, make it appealing to students as well as a useful tool for interactive learning. However, there are significant barriers to its use, including privacy and concerns about professionalism. Higher quality and greater impact research is required.

Expression of Cathepsins B, D, and G in Microcystic Lymphatic Malformation.

Background: This study investigated the expression and localization of cathepsins B, D, and G in relationship to the embryonic stem cell (ESC)-like population we have previously identified in microcystic lymphatic malformation (mLM). Methods and Results: Immunohistochemical staining demonstrated expression of cathepsins B, D, and G in cervicofacial mLM tissue samples from 11 patients. Immunofluorescence staining of two representative mLM samples showed localization of cathepsins B and D to the OCT4+ and the c-MYC+ cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+/tryptase+ cells within the stroma. Transcript expression of cathepsins B, D, and G was confirmed using reverse transcription quantitative polymerase chain reaction (RT-qPCR; n = 5). Western blotting (n = 3) performed on the mLM tissue samples revealed protein expression of cathepsins B and D, which were demonstrated to be enzymatically active using enzymatic activity assays. Conclusion: This study demonstrated expression of cathepsins B and D by the ESC-like cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+ phenotypic mast cells within the stroma of mLM.

Cancer Stem Cells in Head and Neck Cutaneous Squamous Cell Carcinoma Express Cathepsins.

Cancer stem cell (CSC) subpopulations within moderately differentiated head and neck cutaneous squamous cell carcinoma (MDHNcSCC) express the components of the renin-angiotensin system (RAS). This study investigated the expression of cathepsins B, D, and G, which constitute bypass loops of the RAS, by CSCs in MDHNcSCC. METHODS: Immunohistochemical staining was performed on MDHNcSCC tissue samples from 15 patients to determine the expression of cathepsins B, D, and G. Co-localization of these cathepsins with the embryonic stem cell markers Octamer-binding transcription factor 4 (OCT4) and c-MYC was investigated with immunofluorescence staining. Reverse transcription quantitative polymerase chain reaction was performed on 5 MDHNcSCC tissue samples to investigate transcript expression of cathepsins B, D and G. Western blotting and enzymatic activity assays were performed on 5 MDHNcSCC tissue samples and 6 MDHNcSCC-derived primary cell lines to confirm protein expression, transcript expression, and functional activity of these cathepsins, respectively. RESULTS: Immunohistochemical staining demonstrated the expression of cathepsins B, D, and G in all MDHNcSCC tissue samples. Immunofluorescence staining showed localization of cathepsins B and D to the c-MYC+ CSC subpopulations and the OCT4+ CSC subpopulations within the tumor nests and the peritumoral stroma. Cathepsin G was expressed on the tryptase+/c-MYC+ cells within the peritumoral stroma. Reverse transcription quantitative polymerase chain reaction demonstrated transcript expression of cathepsins B, D and G in the MDHNcSCC tissue samples. Western blotting and enzymatic activity assays confirmed protein expression and functional activity of cathepsins B and D in the MDHNcSCC tissue samples and MDHNcSCC-derived primary cell lines, respectively. CONCLUSIONS: Cathepsins B, D, and G are expressed in MDHNcSCC with functionally active cathepsins B and D localizing to the CSC subpopulations, and cathepsin G is expressed by mast cells, suggesting the potential use of cathepsin inhibitors in addition to RAS blockade to target CSCs in MDHNcSCC.

Cancer Stem Cell Subpopulations Are Present Within Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

Cancer stem cells (CSCs) have been identified in many cancer types including primary head and neck cutaneous squamous cell carcinoma (HNcSCC). This study aimed to identify and characterize CSCs in metastatic HNcSCC (mHNcSCC). Immunohistochemical staining performed on mHNcSCC samples from 15 patients demonstrated expression of the induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC in all 15 samples. In situ hybridization and RT-qPCR performed on four of these mHNcSCC tissue samples confirmed transcript expression of all five iPSC markers. Immunofluorescence staining performed on three of these mHNcSCC samples demonstrated expression of c-MYC on cells within the tumor nests (TNs) and the peri-tumoral stroma (PTS) that also expressed KLF4. OCT4 was expressed on the SOX2+/NANOG+/KLF4+ cells within the TNs, and the SOX2+/NANOG+/KLF4+ cells within the PTS. RT-qPCR demonstrated transcript expression of all five iPSC markers in all three mHNcSCC-derived primary cell lines, except for SOX2 in one cell line. Western blotting showed the presence of SOX2, KLF4, and c-MYC but not OCT4 and NANOG in the three mHNcSCC-derived primary cell lines. All three cell lines formed tumorspheres, at the first passage. We demonstrated an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ CSC subpopulation and an OCT4+/NANOG-/SOX2+/KLF4+/c-MYC+ subpopulation within the TNs, and an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ subpopulation within the PTS of mHNcSCC.

Immune Checkpoint Inhibitors in Oral Cavity Squamous Cell Carcinoma and Oral Potentially Malignant Disorders: A Systematic Review.

Cancers of the oral cavity cause significant cancer-related death worldwide. While survival rates have improved in recent years, new methods of treatment are being investigated to limit disease progression and to improve outcomes, particularly in oral cavity squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD). The emerging treatment modality of immunotherapy targets immune checkpoint molecules including PD-1 and its ligand PD-L1, CTLA-4, LAG-3, and TIM-3 to enhance the host immune response against tumours, and to limit the growth and progression of cancer cells. In this systematic review, we searched five databases for keywords pertaining to oral cancers and OPMDs, along with immune checkpoint inhibitors, in order to summarize the current status of their use and efficacy in these diseases. A total of 644 different articles were identified between 2004 and 2019, with 76 deemed suitable for inclusion in the study, providing a total of 8826 samples. Combined results show expression of PD-1 and PD-L1 in the majority of OPMD and OSCC samples, with expression correlating with increased progression and decreased survival rates. Immunotherapy agents pembrolizumab and nivolumab target PD-1 and have been shown to prolong survival rates and improve disease outcomes, especially in combination with chemotherapy or radiotherapy. Despite the equivocal nature of current evidence, there is support for the prognostic and predictive value of immune checkpoint molecules, especially PD-L1, and many studies provide support for the effective use of immune checkpoint inhibitors in the management of OSCC. Limited data is available for OPMD, therefore this should be the focus of future research.

Proliferating Infantile Hemangioma Tissues and Primary Cell Lines Express Markers Associated with Endothelial-to-Mesenchymal Transition.

BACKGROUND: We have previously shown that the endothelium of the microvessels of infantile hemangioma (IH) exhibits a hemogenic endothelium phenotype and proposed its potential to give rise to mesenchymal stem cells, similar to the development of hematopoietic cells. This endothelial-to-mesenchymal transition (Endo-MT) process involves the acquisition of a migratory phenotype by the endothelial cells, similar to epithelial-to-mesenchymal transition that occurs during neural crest development. We hypothesized that proliferating IH expresses Endo-MT-associated proteins and investigated their expression at the mRNA, protein, and functional levels. METHODS: Immunohistochemical staining of paraffin-embedded sections of proliferating IH samples from 10 patients was undertaken to investigate the expression of the Endo-MT proteins Twist1, Twist2, Snail1, and Slug. Transcriptional analysis was performed for the same markers on proliferating IH tissues and CD34+ and CD34- cells from proliferating IH-derived primary cell lines. Adipogenic and osteogenic differentiation plasticity was determined on the CD34-sorted fractions. RESULTS: The endothelium of the microvessels and the cells within the interstitium of proliferating IH tissues expressed Twist1, Twist2, and Slug proteins. Twist1 was also expressed on the pericyte layer of the microvessels, whereas Snail1 was not expressed. Both CD34+ and CD34- populations from the IH-derived primary cell lines underwent adipogenic and osteogenic differentiation. CONCLUSIONS: The expression of Endo-MT-associated proteins Twist1, Twist2, and Slug by both the endothelium of the microvessels and cells within the interstitium, and Twist1 on the pericyte layer of the microvessels of proliferating IH, suggest the presence of a process similar to Endo-MT. This may enable a tightly controlled primitive endothelium of proliferating IH to acquire a migratory mesenchymal phenotype with the ability to migrate away, providing a plausible explanation for the development of a fibrofatty residuum observed during involution of IH.

Identification of Cancer Stem Cell Subpopulations in Head and Neck Metastatic Malignant Melanoma.

Cancer stem cells (CSCs) have been identified in many cancer types. This study identified and characterized CSCs in head and neck metastatic malignant melanoma (HNmMM) to regional lymph nodes using induced pluripotent stem cell (iPSC) markers. Immunohistochemical (IHC) staining performed on 20 HNmMM tissue samples demonstrated expression of iPSC markers OCT4, SOX2, KLF4, and c-MYC in all samples, while NANOG was expressed at low levels in two samples. Immunofluorescence (IF) staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ CSC subpopulation within the tumor nests (TNs) and another within the peritumoral stroma (PTS) of HNmMM tissues. IF also showed expression of NANOG by some OCT4+/SOX2+/KLF4+/c-MYC+ cells within the TNs in an HNmMM tissue sample that expressed NANOG on IHC staining. In situ hybridization (n = 6) and reverse-transcription quantitative polymerase chain reaction (n = 5) on the HNmMM samples confirmed expression of all five iPSC markers. Western blotting of primary cell lines derived from four of the 20 HNmMM tissue samples showed expression of SOX2, KLF4, and c-MYC but not OCT4 and NANOG, and three of these cell lines formed tumorspheres in vitro. We demonstrate the presence of two putative CSC subpopulations within HNmMM, which may be a novel therapeutic target in the treatment of this aggressive cancer.

Expression of Cathepsins B, D, and G by the Embryonic Stem Cell-Like Population within Human Keloid Tissues and Keloid-Derived Primary Cell Lines.

BACKGROUND: The authors have previously shown that an embryonic stem cell-like population within keloid-associated lymphoid tissues in keloid lesions expresses components of the renin-angiotensin system that may be dysregulated. The authors hypothesized that cathepsins B, D, and G are present within the embryonic stem cell-like population in keloid lesions and contribute to bypass loops of the renin-angiotensin system. METHODS: 3,3'-Diaminobenzidine immunohistochemical staining for cathepsins B, D, and G was performed on formalin-fixed paraffin-embedded sections in keloid tissue samples of 11 patients. Immunofluorescence immunohistochemical staining was performed on three of these keloid tissue samples, by co-staining with CD34, tryptase, and OCT4. Western blotting, reverse transcription quantitative polymerase chain reaction, and enzyme activity assays were performed on five keloid tissue samples and four keloid-derived primary cell lines to investigate protein and mRNA expression, and functional activity, respectively. RESULTS: 3,3'-Diaminobenzidine immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 15 keloid tissue samples. Immunofluorescence immunohistochemical staining showed localization of cathepsins B and D to the endothelium of microvessels within the keloid-associated lymphoid tissues and localization of cathepsin G to the tryptase-positive perivascular cells. Western blotting confirmed semiquantitative levels of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines. Reverse transcription quantitative polymerase chain reaction showed quantitative transcriptional activation of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines and cathepsin G in keloid tissue samples. Enzyme activity assays demonstrated functional activity of cathepsins B and D. CONCLUSION: Cathepsins B, D, and G are expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues of keloid lesions and may act to bypass the renin-angiotensin system, suggesting a potential therapeutic target using renin-angiotensin system modulators and cathepsin inhibitors.

Expression of Components of the Renin-Angiotensin System by the Embryonic Stem Cell-Like Population within Keloid Lesions.

BACKGROUND: We investigated expression of prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 by the embryonic stem cell-like population on the endothelium of the microvessels and perivascular cells within keloid-associated lymphoid tissues. METHODS: Immunohistochemical staining for prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 was performed on 11 formalin-fixed, paraffin-embedded sections of keloid tissue samples. Immunofluorescence staining was performed on three keloid tissue samples by co-staining with OCT4, CD34, ERG, and tryptase. Real-time quantitative polymerase chain reaction was performed on five keloid tissue samples and four keloid-derived primary cell lines. Western blotting was performed on the four keloid-derived primary cell lines for mRNA and protein expression of these proteins, respectively. RESULTS: Immunohistochemical and immunofluorescence staining showed expression of prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 in all 11 keloid tissue samples. Prorenin receptor and angiotensin II receptor 1 were expressed on the endothelium and the pericyte layer of the microvessels and perivascular cells, angiotensin II receptor 2 was localized to the endothelium of the microvessels and the tryptase-positive perivascular cells, and angiotensin-converting enzyme was localized to the endothelium of the microvessel, within the keloid-associated lymphoid tissues. Real-time quantitative polymerase chain reaction showed transcripts of prorenin receptor, angiotensin-converting enzyme, and angiotensin II receptor 1 in the keloid tissue samples and keloid-derived primary cell lines, whereas angiotensin II receptor 2 was detected in keloid tissue samples only. Western blotting confirmed the presence of prorenin receptor, angiotensin-converting enzyme, and angiotensin II receptor 1 in the keloid-derived primary cell lines. CONCLUSION: Prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 were expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues, suggesting that this primitive population may be a potential therapeutic target by modulation of the renin-angiotensin system.

Expression of Components of the Renin-Angiotensin System by the Putative Stem Cell Population Within WHO Grade I Meningioma.

Aim: We have recently demonstrated a putative stem cell population within WHO grade I meningioma (MG) that expressed embryonic stem cell (ESC) markers OCT4, NANOG, SOX2, KLF4 and c-MYC, localized to the endothelial and pericyte layers of the microvessels. There is increasing recognition that the renin-angiotensin system (RAS) plays a critical role in stem cell biology and tumorigenesis. This study investigated the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on the putative stem cell population on the microvessels of WHO grade I MG. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on WHO grade I MG tissue samples from 11 patients for PRR, ACE, ATIIR1, and ATIIR2. Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these components of the RAS in using combinations of CD34 and ESC marker SOX2 or OCT4. NanoString mRNA expression analysis and Western blotting (WB), were performed on six snap-frozen MG tissue samples to confirm mRNA and protein expression of these proteins, respectively. Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 within all 11 MG tissue samples. WB and NanoString mRNA analyses, confirmed protein and mRNA expression of these proteins, respectively. IF IHC staining showed PRR, ATIIR1 and ATIIR2 were localized to the OCT4+ and SOX2+ endothelium and the pericyte layer of MG while ACE was localized to the OCT4+ endothelium of the microvesels. Conclusion: The novel finding of the expression of PRR, ACE, ATIIR1, and ATIIR2 on the putative stem cell population on the microvessels of WHO grade I MG, suggests that these stem cells may be a potential therapeutic target by manipulation of the RAS.

Expression of (pro)renin receptor and its effect on endothelial cell proliferation in infantile hemangioma.

BACKGROUND: Propranolol is the preferred treatment for problematic proliferating infantile hemangioma (IH) by targeting the renin-angiotensin system (RAS) expressed by IH endothelium. (Pro)renin receptor (PRR) is a major component of the RAS associated with the canonical wnt signaling pathway. We proposed that activation of PRR by renin causes proliferation of IH. METHODS: The expression of PRR in IH tissue samples was investigated using immunohistochemical (IHC) staining and NanoString analysis. NanoString analysis was also used to confirm transcriptional expression of PRR in CD34-sorted proliferating IH-derived primary cell lines. MTT assay was utilized to determine the effect of exogenous renin on the number of viable IH cells. RT-qPCR was used to determine the effect of renin on the stem cell gene expression. RESULTS: NanoString analysis and IHC staining confirmed transcriptional and translational expression of PRR, which was localized to the non-endothelial and the endothelial IH cell populations. MTT assay demonstrated an increased number of viable IH cells by administration of renin and the effect was negated by the wnt receptor blocker dickkopf-1. CONCLUSION: Our results present a model for renin-induced increased proliferation of IH cells through PRR acting via the wnt signaling pathway, which may account for accumulation of cells in IH during the proliferative phase of the tumor.

Expression of Cathepsins B, D, and G in WHO Grade I Meningioma.

Aim: We have recently demonstrated the presence of putative tumor stem cells (TSCs) in World Health Organization (WHO) grade I meningioma (MG) localized to the microvessels, which expresses components of the renin-angiotensin system (RAS). The RAS is known to be dysregulated and promotes tumorigenesis in many cancer types, including glioblastoma. Cathepsins B, D, and G are isoenzymes that catalyze the production of angiotensin peptides, hence providing bypass loops for the RAS. This study investigated the expression of cathepsins B, D, and G in WHO grade I MG in relation to the putative TSC population we have previously demonstrated. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining with antibodies for cathepsins B, D, and G was performed on WHO grade I MG tissue samples from 10 patients. Three of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these cathepsins using combinations of smooth muscle actin (SMA) and embryonic stem cell marker OCT4. NanoString mRNA expression (n = 6) and Western blotting (WB; n = 5) analyses, and enzyme activity assays (EAAs; n = 3), were performed on snap-frozen WHO grade I MG tissue samples to confirm transcriptional activation, protein expression, and functional activity of these proteins, respectively. Results: DAB IHC staining demonstrated expression of cathepsins B, D, and G in all 10 MG samples. NanoString mRNA expression and WB analyses showed transcriptional activation and protein expression of all three cathepsins, although cathepsin G was expressed at low levels. EAAs demonstrated that cathepsin B and cathepsin D were functionally active. IF IHC staining illustrated localization of cathepsin B and cathepsin D to the endothelium and SMA+ pericyte layer of the microvessels, while cathepsin G was localized to cells scattered within the interstitium, away from the microvessels. Conclusion: Cathepsin B and cathepsin D, and to a lesser extent cathepsin G, are expressed in WHO grade I MG. Cathepsin B and cathepsin D are enzymatically active and are localized to the putative TSC population on the microvessels, whereas cathepsin G was localized to cells scattered within the interstitium, These results suggest the presence of bypass loops for the RAS, within WHO grade I MG.

Expression of Components of the Renin-Angiotensin System in Pyogenic Granuloma.

Background: There is a growing body of research demonstrating expression of the renin-angiotensin system (RAS) by a putative embryonic stem cell (ESC)-like population within vascular anomalies. This study investigated the expression of components of the RAS in relation to the putative ESC-like population within pyogenic granuloma (PG) that we have recently reported. Methods: PG samples from 14 patients were analyzed for the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), using 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining. Immunofluorescence (IF) IHC staining was performed to localize these proteins on four of the PG samples. RT-qPCR was performed on two snap-frozen PG samples. Western blotting (WB) was performed on one snap-frozen PG sample and two PG-derived primary cell lines. Results: DAB IHC staining demonstrated the expression of ACE, PRR, ATIIR1, and ATIIR2 in all 14 PG tissue samples. RT-qPCR analysis confirmed abundant mRNA transcripts for PRR, ACE, AIITR1 and ATIIR2, relative to the housekeeping gene. WB confirmed the presence of PRR, ATIIR1, and ACE in the PG tissue sample, and PRR and ATIIR1, in the PG-derived primary cell lines. IF IHC staining demonstrated the expression of PRR, ACE, ATIIR1 on the primitive population that expressed NANOG and SOX2 on the ERG+ endothelium of the microvessels within PG. Conclusion: We have demonstrated the expression of PRR, ACE, and ATIIR1 by the putative the ESC-like population within PG.

Circulating tumor stem cells and glioblastoma: A review.

Glioblastoma (GB) is the most aggressive primary brain tumor in adults. The aggressive nature of GB has been attributed to the presence of cancer stem cells (CSCs) which drive tumorigenesis and are thought to be the root cause of the disease. Circulating tumor stem cells (CTSCs), which can be derived from CSCs, have been identified in numerous types of cancer including GB, have been proposed to contribute to local and distant recurrence. There are many technical difficulties in studying CTSCs, therefore there is a significant gap in the literature pertaining to how they arise and function, and how the understanding of the biology of CTSCs could elucidate the underlying cause of local recurrence and metastasis. An initial epithelial-to-mesenchymal transition (EMT) followed by mesenchymal-to-epithelial transition involving these primitive cells appear to be the critical processes underpinning metastasis. This review focuses on the association between CSCs undergoing EMT to become CTSCs, and how this could arise from the CSC subpopulation in GB, and contribute to the understanding of the pathogenesis and treatment.

Keloid-associated lymphoid tissues in keloid lesions express vitamin D receptor.

Objectives: Vitamin D receptor (VDR) may play a role in keloid disorder. This study investigated the expression of VDR by the embryonic stem cell (ESC)-like population within keloid-associated lymphoid tissues (KALTs) which expresses components of the renin-angiotensin system (RAS). Methods: 11 formalin-fixed paraffin-embedded sections of keloid lesions (KLs) underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for VDR. Immunofluorescence (IF) dual IHC staining of CD34/VDR and OCT4/VDR was performed on two representative KLs. Transcriptional activation of VDR was investigated in four representative snap-frozen KLs using reverse-transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: DAB IHC staining demonstrated the presence of VDR on the KALTs within the keloid tissue samples. RT-qPCR confirmed transcriptional activation of VDR. IF IHC staining demonstrated expression of VDR on the CD34+ and the OCT4+ endothelium of the microvessels, and the OCT4+ perivascular cells, within the KALTs. Conclusions: This study demonstrated the expression of VDR by the ESC-like population within the KALTs in KLs. Further work is needed to elucidate the precise interaction between VDR and the RAS in regulating the primitive population within the KALTs.

Expression and Localization of Cathepsins B, D and G in Cancer Stem Cells in Liver Metastasis From Colon Adenocarcinoma.

AIM: We have previously identified and characterized cancer stem cell (CSC) subpopulations in liver metastasis from colon adenocarcinoma (LMCA). In this study we investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. METHODS: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D and G was performed on 4µm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients. Immunofluorescence (IF) IHC staining was performed on three representative samples of LMCA from the original cohort of nine patients, to determine the localization of these cathepsins in relation to the CSC subpopulations. NanoString mRNA analysis and Western Blotting (WB) were used to examine the transcript and protein expression of these cathepsins, respectively. Enzyme activity assays were utilized to determine their functional activity. Data acquired from counting of cells staining positively of the cathepsins on the DAB IHC-stained slides and from Nanostring mRNA analysis were subjected to statistical analyses to determine significance. RESULTS: DAB IHC staining demonstrated expression of cathepsins B, D and G within LMCA. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4- cells within the tumor nests and the OCT4+ CSC subpopulation within the peritumoral stroma. NanoString mRNA analysis showed significantly greater transcript expression of cathepsin B and cathepsin D, compared to cathepsin G. WB confirmed expression of cathepsin B and cathepsin D proteins, while cathepsin G was below detectable levels. Enzyme activity assays showed functional activity of cathepsin B and cathepsin D. CONCLUSION: Our study demonstrated novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA.