Neuroepithelial Origin Research Articles

Histopathologic and molecular profile of gliomas diagnosed in Lagos, Nigeria

Abstract Background The optimal diagnosis and management of patients with brain tumors currently uses the 2021 WHO integrated diagnosis of histomorphologic and molecular features. However, neuro-oncology practice in resource-limited settings usually relies solely on histomorphology. This study aimed to classify glioma cases diagnosed in the Department of Anatomic and Molecular Pathology, Lagos University Teaching Hospital, using the 2021 WHO CNS tumor classification. Methods Fifty-six brain tumors from 55 patients diagnosed with glioma between 2013 and 2021 were reevaluated for morphologic diagnosis. Molecular features were determined from formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) for IDH1-R132H, ATRX, BRAF-V600E, p53, Ki67, and H3-K27M, OncoScan chromosomal microarray for copy number, targeted next generation sequencing for mutation and fusion and methylation array profiling. Results Of 55 central nervous system tumors, 3 were excluded from histomorphologic reevaluation for not being of glial or neuroepithelial origin. Of the remaining 52 patients, the median age was 20.5 years (range: 1 to 60 years), 38(73%) were males and 14(27%) were females. Seventy-one percent of the gliomas evaluated provided adequate DNA from archival FFPE tissue blocks. After applying the 2021 WHO diagnostic criteria the initial morphologic diagnosis changed for 35% (18/52) of cases. Diagnoses of 5 (9.6%) gliomas were upgraded, and 7 (14%) were downgraded. Conclusions This study shows that the incorporation of molecular testing can considerably improve brain tumor diagnoses in Nigeria. Furthermore, this study highlights the diagnostic challenges in resource-limited settings and what is at stake in the global disparities of brain tumor diagnosis.

Intracerebral retina-like pigmented tissue in a stillborn fetus with holoprosencephaly

A stillbirth fetus with semilobar holoprosencephaly was induced at 24 weeks gestational age. While the eyes appeared unremarkable externally, there was an absence of optic nerves. At the ventral hypothalamicdiencephalic region there was an area of bilateral epithelioid cells containing melanin. Immunohistochemical characterization revealed the cells to be of neuroepithelial origin with features of retinal pigment epithelium. These findings reflect abnormalities in eye development in holoprosencephaly, especially when coupled with other structural defects in the visual system.

Alternative Lengthening of Telomeres and Mediated Telomere Synthesis.

Simple SummaryAlternative lengthing of telomere (ALT) is an important mechanism for maintaining telomere length and cell proliferation in telomerase-negative tumor cells. However, the molecular mechanism of ALT is still poorly understood. ALT occurs in a wide range of tumor types and usually associated with a worse clinical consequence. Here, we review the recent findings of ALT mechanisms, which promise ALT could be a valuable drug target for clinical telomerase-negative tumor treatment.Telomeres are DNA–protein complexes that protect eukaryotic chromosome ends from being erroneously repaired by the DNA damage repair system, and the length of telomeres indicates the replicative potential of the cell. Telomeres shorten during each division of the cell, resulting in telomeric damage and replicative senescence. Tumor cells tend to ensure cell proliferation potential and genomic stability by activating telomere maintenance mechanisms (TMMs) for telomere lengthening. The alternative lengthening of telomeres (ALT) pathway is the most frequently activated TMM in tumors of mesenchymal and neuroepithelial origin, and ALT also frequently occurs during experimental cellular immortalization of mesenchymal cells. ALT is a process that relies on homologous recombination (HR) to elongate telomeres. However, some processes in the ALT mechanism remain poorly understood. Here, we review the most recent understanding of ALT mechanisms and processes, which may help us to better understand how the ALT pathway is activated in cancer cells and determine the potential therapeutic targets in ALT pathway-stabilized tumors.

Spectrum of Central Nervous System Tumours at Tertiary Care Centre in Nepal

IntroductionMost of the Central Nervous System tumors are benign. There is increase in the incidence in recent days which might be due to improved diagnosis with advancement in the ancillary studies. This study aims to provide a single centre histopathological spectrum of tumor of CNS. MethodsThis is a retrospective cross-sectional study of 138 cases of CNS tumors managed surgically in the department of neurosurgery from 2017 to 2020.All the histological subtypes and grading were reviewed. Data of the study population were analysed using SPSS version 25 and Microsoft excel. All the patients were classified as per the 2007 World Health Organization classification of central nervous system tumors. ResultsA total of 138 cases were analysed.One hundred and thirteen constitutes brain tumors and remaining 25 constitute the spinal tumors. Of the total cases, 132 were primary CNS tumor and remaining six were secondary CNS tumor. In cranial, 61 (44.2%) were extra axial tumors. There were 122 low grade tumor and remaining 16 were high grade tumors. Tumors of neuroepithelial origin were the most common 54 (38.9%) CNS tumors followed by meningiothelial tumors 36 (26.0%).Tumor were equally distributed in both the sex.Mean age of study population was 37.38 years ranging from 0.5 years to 75 years. ConclusionsThe present study concludes that the most common CNS tumour at our centre were neuroepithelial tumor mainly pilocytic astrocytoma followed by meningothelial.

Exploring the Pivotal Neurophysiologic and Therapeutic Potentials of Vitamin C in Glioma

Gliomas represent solely primary brain cancers of glial cell or neuroepithelial origin. Gliomas are still the most lethal human cancers despite modern innovations in both diagnostic techniques as well as therapeutic regimes. Gliomas have the lowest overall survival rate compared to other cancers 5 years after definitive diagnosis. The dietary intake of vitamin C has protective effect on glioma risk. Vitamin C is an essential compound that plays a vital role in the regulation of lysyl and prolyl hydroxylase activity. Neurons store high levels of vitamin C via sodium dependent-vitamin C transporters (SVCTs) to protect them from oxidative ischemia-reperfusion injury. Vitamin C is a water-soluble enzyme, typically seen as a powerful antioxidant in plants as well as animals. The key function of vitamin C is the inhibition of redox imbalance from reactive oxygen species produced via the stimulation of glutamate receptors. Gliomas absorb vitamin C primarily via its oxidized dehydroascorbate form by means of GLUT 1, 3, and 4 and its reduced form, ascorbate, by SVCT2. Vitamin C is able to preserve prosthetic metal ions like Fe2+ and Cu+ in their reduced forms in several enzymatic reactions as well as scavenge free radicals in order to safeguard tissues from oxidative damage. Therapeutic concentrations of vitamin C are able to trigger H2O2 generation in glioma. High-dose combination of vitamin C and radiation has a much more profound cytotoxic effect on primary glioblastoma multiforme cells compared to normal astrocytes. Control trials are needed to validate the use of vitamin C and standardization of the doses of vitamin C in the treatment of patients with glioma.

Astroglia in ageing

Astroglia are neural cells of ectodermal, neuroepithelial origin responsible for homoeostasis and defence of the central nervous system (CNS). Ageing reduces the functional capacity of all organs, so does that of the nervous system, the latter is evident in the reduction of cognitive abilities, learning and memory. At the same time the progression of these deficits is very much individual and lifestyle dependent, indicating operation of mechanisms counterbalancing age-dependent decline. In physiological ageing astrocytes undergo morphological atrophy and functional asthenia; astrocytic paralysis facilitates progression of age-dependent neurodegenerative disorders. Astroglial status and homoeostatic capabilities are influenced by lifestyle including intellectual engagement, social interactions, physical exercise, and healthy diet. Maintenance of healthy lifestyle is paramount for cognitive longevity.

Rare germline variants in the E-cadherin gene CDH1 are associated with the risk of brain tumors of neuroepithelial and epithelial origin

The genetic basis of brain tumor development is poorly understood. Here, leukocyte DNA of 21 patients from 15 families with ≥ 2 glioma cases each was analyzed by whole-genome or targeted sequencing. As a result, we identified two families with rare germline variants, p.(A592T) or p.(A817V), in the E-cadherin gene CDH1 that co-segregate with the tumor phenotype, consisting primarily of oligodendrogliomas, WHO grade II/III, IDH-mutant, 1p/19q-codeleted (ODs). Rare CDH1 variants, previously shown to predispose to gastric and breast cancer, were significantly overrepresented in these glioma families (13.3%) versus controls (1.7%). In 68 individuals from 28 gastric cancer families with pathogenic CDH1 germline variants, brain tumors, including a pituitary adenoma, were observed in three cases (4.4%), a significantly higher prevalence than in the general population (0.2%). Furthermore, rare CDH1 variants were identified in tumor DNA of 6/99 (6%) ODs. CDH1 expression was detected in undifferentiated and differentiating oligodendroglial cells isolated from rat brain. Functional studies using CRISPR/Cas9-mediated knock-in or stably transfected cell models demonstrated that the identified CDH1 germline variants affect cell membrane expression, cell migration and aggregation. E-cadherin ectodomain containing variant p.(A592T) had an increased intramolecular flexibility in a molecular dynamics simulation model. E-cadherin harboring intracellular variant p.(A817V) showed reduced β-catenin binding resulting in increased cytosolic and nuclear β-catenin levels reverted by treatment with the MAPK interacting serine/threonine kinase 1 inhibitor CGP 57380. Our data provide evidence for a role of deactivating CDH1 variants in the risk and tumorigenesis of neuroepithelial and epithelial brain tumors, particularly ODs, possibly via WNT/β-catenin signaling.

Astrocytes: The Housekeepers and Guardians of the CNS.

Astroglia are a diverse group of cells in the central nervous system. They are of the ectodermal, neuroepithelial origin and vary in morphology and function, yet, they can be collectively defined as cells having principle function to maintain homeostasis of the central nervous system at all levels of organisation, including homeostasis of ions, pH and neurotransmitters; supplying neurones with metabolic substrates; supporting oligodendrocytes and axons; regulating synaptogenesis, neurogenesis, and formation and maintenance of the blood-brain barrier; contributing to operation of the glymphatic system; and regulation of systemic homeostasis being central chemosensors for oxygen, CO2 and Na+. Their basic physiological features show a lack of electrical excitability (inapt to produce action potentials), but display instead a rather active excitability based on variations in cytosolic concentrations of Ca2+ and Na+. It is expression of neurotransmitter receptors, pumps and transporters at their plasmalemma, along with transports on the endoplasmic reticulum and mitochondria that exquisitely regulate the cytosolic levels of these ions, the fluctuation of which underlies most, if not all, astroglial homeostatic functions.

PATH-42. COMBINED NEXT GENERATION SEQUENCING AND IMMUNOHISTOCHEMISTRY IN THE DIFFERENTIAL DIAGNOSIS OF A PROGRESSIVE NEURONAL-GLIAL TUMOR: A COMPOSITE NEOPLASM

Abstract BACKGROUND Neuronal-Glial tumors (NGT) are rare neoplasms typically benign, and characterized by various admixtures of neuronal and glial components. Occasionally these tumors transform to higher grades and rarely develop other histologic features inconsistent with a neuroepithelial origin, raising the question of histogenesis. We present a case of World Health Organization (WHO) grade I ganglioglioma (GG) diagnosed in a 28 year old man whose tumor, over a period of 8 years, progressed to anaplastic ganglioglioma (AGG, WHO grade III) and ultimately AGG with high grade sarcomatoid features. The sarcomatoid component eventually outgrew the NGT components, resulting in the patient’s death. We used next generation sequencing (NGS) and immunohistochemistry to characterize the genetic changes associated with these events. METHODS Tissues representing NGT and sarcomatoid components were macrodissected from 6 samples representing the tumor during its evolution from low to higher grades. DNA and RNA were extracted from these samples and NGS performed using the Oncomine Comprehensive Panel. Whole exome sequencing was performed using DNA from the 6 tumor samples and adjacent non-neoplastic tissue. RESULTS The original tumor, WHO grade I ganglioglioma had a BRAF V600E mutation. Seven years later, the AGG component had the BRAF V600E, absent in the sarcomatoid component, which had a CDKN2A homozygous deletion. The following year, the sarcomatoid component appeared to predominate and had an additional PIK3CA activating mutation. The NGT component retained the original BRAF mutation but no others. Hierarchical clustering of genome-wide variants identified two distinct cell populations emerging over time: GG progressing to AGG and sarcomatoid tumor. CONCLUSIONS Based on these results, we propose two distinct tumor cell populations: GG/AGG and sarcomatoid component, evolving over time and resulting in what is commonly considered a collision tumor, two independent tumors adjacent to each other.

Primary primitive neuroectodermal tumor of the orbit in an adult: Case report and literature review

Background: Primitive neuroectodermal tumors (PNETs) are malignant soft tissue tumors of neuroepithelial origin with an aggressive nature and associated with early disseminated metastasis, resulting in poor prognosis. It occurs more frequently in men of age < 35 years, and in several parts of the body, such as pulmonary tract, nasal cavity, paranasal sinuses, and/or neck. Orbital location is infrequent and has been reported in less than 20 cases in the English literature so far.
 Case presentation: In this paper, we report our experience with a 77 year-old man who was referred with a 2.8 cm mass in the superior-anterior quadrant of his left eye for 2 weeks prior to admission, as well as having a 15-kg weight loss during the past six months. The patient developed rapidly progressive visual loss, decreased eye movements, and conjunctivitis within a week. Thus, the patient underwent orbitotomy and the results of histological examination and immunohistochemistry showed an orbital PNET. Metastasis to frontal sinuses and ethmoid sinuses were also discovered. The patient passed away before referral to the oncologist.
 Conclusion: This report confirms the highly aggressive nature of PNET and reports its rare occurrence in orbital cavity.

Pak1ip1 Loss-of-Function Leads to Cell Cycle Arrest, Loss of Neural Crest Cells, and Craniofacial Abnormalities.

Neural crest cells (NCCs) comprise a transient progenitor cell population of neuroepithelial origin that contributes to a variety of cell types throughout vertebrate embryos including most mesenchymal cells of the cranial and facial structures. Consequently, abnormal NCC development underlies a variety of craniofacial defects including orofacial clefts, which constitute some of the most common birth defects. We previously reported the generation of manta ray (mray) mice that carry a loss-of-function allele of the gene encoding the preribosomal factor Pak1ip1. Here we describe cranioskeletal abnormalities in homozygous mray mutants that arise from a loss of NCCs after their specification. Our results show that the localized loss of cranial NCCs in the developing frontonasal prominences is caused by cell cycle arrest and cell death. In addition, and consistent with deficits in ribosome biosynthesis, homozygous mray mutants display decreased protein biosynthesis, further linking Pak1ip1 to a role in ribosome biogenesis.

Apport de la TDM dans le diagnostic et le suivi du retinoblastome au Sénégal

Retinoblastoma is a malignant tumor of neuroepithelial origin, developed from young retinal cells, occurring in infants and young children. The goal of the study was to assess the role of CT in the diagnosis of retinoblastoma at the Aristide le Dantec Hospital in Dakar. This is an 11-year retrospective study of 160 patient records in the ophthalmology department and pediatric oncology unit of the same hospital. One hundred and sixty (160) patients were recruited. The male:female ratio was 1.05. The mean age was 32.19 months. Leukocoria was the most common clinical sign, occurring in 105 cases (65.62%). The retinoblastoma was intraocular in 97 cases (60.62%). We saw 122 (76.25%) unilateral and 3 (1.87%) trilateral presentations. CT was performed in 150 children with 110 cases (73.33%) of calcifications; 62 cases (41.33%) of optic nerve invasion; 24 cases (16%) of extraocular muscle invasion and 18 cases (12%) endocranial extension. Associated tumors were found on CT: 2 cases of suprasellar mass and 1 case of pineoblastoma. In patients whose specimens were analyzed, histology showed 48.15% optic nerve invasion, consistent with the CT findings. CT has an important role in the diagnosis of retinoblastoma, despite its recent contraindication in bilateral and unilateral multinodular forms. CT is a good alternative to MRI in the diagnosis of retinoblastoma in developing countries with limited technological resources.

Expression of Insulinoma-Associated Protein 1 (INSM1) and Orthopedia Homeobox (OTP) in Tumors with Neuroendocrine Differentiation at Rare Sites.

Insulinoma-associated protein 1 (INSM1) and orthopedia homeobox (OTP) are transcription factors that play a critical role in neuroendocrine (NE) and neuroepithelial cell development. INSM1 has been identified in multiple tumors of NE or neuroepithelial origin, whereas OTP expression has been mainly studied in NE tumors of pulmonary origin. Expression of OTP appears to correlate with poorer prognosis in pulmonary carcinoids; however, its expression patterns in other NE/neuroepithelial tumors need further investigation. Here, we assessed the diagnostic utility of INSM1 and OTP in tumors with NE differentiation at relatively uncommon sites including prostate, breast, and tumors of gynecologic origin. Thirty-two formalin-fixed, paraffin-embedded cases were used to construct a tissue microarray. Immunohistochemistry for INSM1 and OTP was performed and scored semi-quantitatively. INSM1 was diffusely expressed in 60% of gynecologic tumors, 71.4% of mammary carcinoma, and 25% of prostate adenocarcinoma with NE differentiation. Diffuse expression of OTP was detected in 50% of prostate adenocarcinoma with NE differentiation and 100% neuroendocrine carcinoma of the ovary. Immunostain for achaete-scute homolog 1, chromogranin, synaptophysin, and CD56 supported the NE and/or neuroepithelial differentiation of the tumors. In summary, INSM1 is expressed in most of the tumors with NE and neuroepithelial differentiation in this study, confirming the diagnostic utility of INSM1 as a novel and sensitive marker of NE/neuroepithelial differentiation. The expression of OTP in some NE tumors outside of lung expands the spectrum of tumors that may express this biomarker and should be considered when working up a NE tumor of unknown primary site.

Intracranial Germ Cell Tumor in the Molecular Era.

Intracranial germ cell tumors (iGCTs) are a heterogeneous group of tumors with peculiar characteristics clearly distinguished from other brain tumors of neuroepithelial origin. Diverse histology, similarity to gonadal GCT, predilection to one sex, and geographic difference in incidence all present enigmas and fascinating challenges. The treatment of iGCT has advanced for germinoma to date; thus, clinical attention has shifted from survival to long-term quality of life. However, for non-germinomatous GCT, current protocols provide only modest improvement and more innovative therapies are needed. Recently, next-generation sequencing studies have revealed the genomic landscape of iGCT. Novel mutations in the KIT-RAS-MAPK and AKT-MTOR pathways were identified. More importantly, methylation profiling revealed a new method to assess the pathogenesis of iGCT. Molecular research will unleash new knowledge on the origin of iGCT and solve the many mysteries that have lingered on this peculiar neoplasm for a long time.

Embryonal Tumors of the Central Nervous System: From the Radiologic Pathology Archives.

Embryonal tumors of the central nervous system (CNS) are highly malignant undifferentiated or poorly differentiated tumors of neuroepithelial origin and have been defined as a category in the World Health Organization (WHO) classification since the first edition of the "Blue Book" in 1979. This category has evolved over time to reflect our ever-improving understanding of tumor biology and behavior. With the most recent update in 2016, many previous histologic diagnoses incorporate molecular parameters for the first time (genetically defined entities). While medulloblastoma and atypical teratoid/rhabdoid tumor are familiar carryovers from the 2007 CNS WHO classification, there are major changes to the embryonal tumor category: for example, elimination of the term CNS primitive neuroectodermal tumor and addition of a new genetically defined entity, embryonal tumor with multilayered rosettes, C19MC-altered. The purpose of this article is to discuss both the radiologic-pathologic features of CNS embryonal tumors and the new molecularly defined types/subtypes that will become the standard classification/terminology for future diagnoses and tumor research.

A Comparative Review of Demographics, Incidence, and Epidemiology of Histologically Confirmed Intracranial Tumors in Brazil and Bulgaria.

Intracranial tumors (ICTs) attract numerous scientific teams and tremendous financial resources worldwide. These lesions of the central nervous system (CNS) can be both benign and malignant in biological behavior as well as local or metastatic in origin. We compared data from two studies on primary and metastatic ICTs from Brazil and Bulgaria, based on histopathologically confirmed ICTs from tertiary health centers. Primary ICTs significantly outweigh the frequency of metastatic ICTs. Primary ICTs represent 86.45% in Brazil and 69.17% in Bulgaria, with around 60% of their totals being malignant. There is a statistical dominance of tumors from the neuroepithelial origin, with the most common entry being glioblastoma multiforme. The second-most common primary ICT group comprises tumors of meningeal origin. Metastatic ICTs show great variance; 13.55% in Brazil and 31.38% in Bulgaria of all ICT cases being attributed to them. However, metastatic ICTs are even a more diverse group than neuroepithelial tumors, with the majority of this group comprising metastatic colorectal adenocarcinoma (almost exclusively in males), metastatic breast adenocarcinoma in females, metastatic pulmonary carcinomas (primarily from the non-small cell group with a male predominance), and metastatic melanoma with an even gender ratio.

Physiology of Astroglia.

Astrocytes are neural cells of ectodermal, neuroepithelial origin that provide for homeostasis and defense of the central nervous system (CNS). Astrocytes are highly heterogeneous in morphological appearance; they express a multitude of receptors, channels, and membrane transporters. This complement underlies their remarkable adaptive plasticity that defines the functional maintenance of the CNS in development and aging. Astrocytes are tightly integrated into neural networks and act within the context of neural tissue; astrocytes control homeostasis of the CNS at all levels of organization from molecular to the whole organ.

Juxtaoral organ of Chievitz: An innocuous organ to be known.

The Juxtaoral Organ of Chievitz is a normal anatomical structure located within the soft tissue in the buccotemporal fascia on the medial surface of the ascending ramus. This enigmatic vestigial structure is considered to be of neuroepithelial origin with no known function. As a matter of fact, JOOC is one of the most treacherous pitfalls in surgical pathology with respect to lesions in the head and neck area. Hence the basic aim of this short communication is to reveal the importance about this organ and enlighten the oral pathologist about this histopathological structure, thus preventing extensive and unnecessary investigations.

INSM1: A Novel Immunohistochemical and Molecular Marker for Neuroendocrine and Neuroepithelial Neoplasms

Neuroendocrine neoplasms (NENs) are heterogeneous neoplasms, which are sometimes malignant, although predicting metastasis is difficult. INSM1 is a transcription factor expressed transiently in embryonic neuroendocrine (NE) tissue, thought to coordinate termination of cell division with differentiation of NE and neuroepithelial cells. In adult tissues, INSM1 has been identified in multiple tumors of NE or neuroepithelial origin but has not been thoroughly investigated as a potential neoplastic marker. We evaluated INSM1 as a semiquantitative immunohistochemical (IHC) marker for NE and neuroepithelial neoplasms and as a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) marker for gastrointestinal NENs (GI-NENs). Using IHC, we found in normal adult tissue that INSM1 expression was highly restricted to nuclei of NE cells and tissues. INSM1 was not detected in any adult nonneoplastic, non-NE tissue. In neoplastic tissue, INSM1 was detectable by IHC in 88.3% of 129 NEN specimens. In contrast, INSM1 was detected by IHC in only one of 27 neoplasms without a neuroepithelial or NE component. Using qRT-PCR, we evaluated INSM1 gene expression in 113 GI-NEN specimens. INSM1 expression was significantly increased in neoplastic vs nonneoplastic tissue. Furthermore, among midgut GI-NENs, neoplasms with known metastases showed significantly higher expression than those that had not yet metastasized.

The effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells.

Recently, it has been shown that silver nanoparticles (AgNPs) provide a unique approach to the treatment of tumors, especially those of neuroepithelial origin. Thus, the aim of this study was to evaluate the impact of AgNPs on proliferation and activation of the intrinsic apoptotic pathway of glioblastoma multiforme (GBM) cells cultured in an in ovo model. Human GBM cells, line U-87, were placed on chicken embryo chorioallantoic membrane. After 8 days, the tumors were divided into three groups: control (non-treated), treated with colloidal AgNPs (40 μg/ml), and placebo (tumors supplemented with vehicle only). At the end of the experiment, all tumors were isolated. Assessment of cell proliferation and cell apoptosis was estimated by histological, immunohistochemical, and Western blot analyses. The results show that AgNPs can influence GBM growth. AgNPs inhibit proliferation of GBM cells and seem to have proapoptotic properties. Although there were statistically significant differences between control and AgNP groups in the AI and the levels of active caspase 9 and active caspase 3, the level of these proteins in GBM cells treated with AgNPs seems to be on the border between the spontaneous apoptosis and the induced. Our results indicate that the antiproliferative properties of silver nanoparticles overwhelm proapoptotic ones. Further research focused on the cytotoxic effect of AgNPs on tumor and normal cells should be conducted.Electronic supplementary materialThe online version of this article (doi:10.1186/s11671-015-0823-5) contains supplementary material, which is available to authorized users.