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Biomedical subjects

D Oda

Publications and source records attributed to D Oda.

At least 19 recordsLinked to original sources

Distinctive characteristics of transcriptional profiles from two epithelial cell lines upon interaction with Actinobacillus actinomycetemcomitans.

Transcriptional profiling and gene ontology analyses were performed to investigate the unique responses of two different epithelial cell lines to an Actinobacillus actinomycetemcomitans challenge. A total of 2867 genes were differentially regulated among all experimental conditions. The analysis of these 2867 genes revealed that the predominant specific response to infection in HeLa cells was associated with the regulation of enzyme activity, RNA metabolism, nucleoside and nucleic acid transport and protein modification. The predominant specific response in immortalized human gingival keratinocytes (IHGK) was associated with the regulation of angiogenesis, chemotaxis, transmembrane receptor protein tyrosine kinase signaling, cell differentiation, apoptosis and response to stress. Of particular interest, stress response genes were significantly - yet differently - affected in both cell lines. In HeLa cells, only three regulated genes impacted the response to stress, and the response to unfolded protein was the only term that passed the ontology filters. This strikingly contrasted with the profiles obtained for IHGK, in which 61 regulated genes impacted the response to stress and constituted an extensive network of cell responses to A. actinomycetemcomitans interaction (response to pathogens, oxidative stress, unfolded proteins, DNA damage, starvation and wounding). Hence, while extensive similarities were found in the transcriptional profiles of these two epithelial cell lines, significant differences were highlighted. These differences were predominantly found in pathways that are associated with host-pathogen interactions.

Aggregatibacter actinomycetemcomitans↗

Orofacial rhabdomyosarcoma in neonates and young children: a review of literature and management of four cases.

Rhabdomyosarcoma (RMS) is an aggressive malignant skeletal muscle neoplasm arising from embryonal mesenchyme. It accounts for over 50% of all pediatric soft tissue sarcomas. The head and neck region is the most common site for this tumor in children. Neonatal presentation of this tumor is rare. We present the management of one neonatal case and three additional cases of orofacial RMS in children under the age of 7 years. All four patients were seen in the department of oral and maxillofacial surgery at Children's Hospital and Regional Medical Center (CHRMC) in Seattle between 1992-2000. Three of the four cases were alveolar RMS and one was botryoid sub-type of embryonal RMS. Three patients were treated with a combination of surgery, chemotherapy and radiation, while the patient with botryoid RMS was treated with surgery and chemotherapy only. The patient with congenital RMS died at 2.5 years of age due to recurrent metastatic disease. The other three patients are alive without evidence of recurrent with a mean follow up was 5.5 years (range 2.5-8.5 years). We discuss the current management, diagnosis, biological behavior, histopathology, prognosis and survival of head and neck RMS in neonates and young children.

Child↗

Odontogenic keratocyst: the northwestern USA experience.

Odontogenic keratocyst (OKC) is a cyst of tooth origin with an aggressive clinical behavior including a high recurrence rate. OKC demographics in the northwestern United States are presented and compared to those reported elsewhere. A total of 430 cases were obtained from 393 patients of the northwest region over a period of 15 years. Data evaluated included: site, gender, age, race, and association with bifid-rib basal cell nevus syndrome (Gorlin syndrome). Site distribution of the northwest group was similar to that of international groups. For the northwest group, the most common lesion location was the body of the mandible. Gender distribution in the northwest group appeared similar to other reports made in Denmark, England, Japan, and other regions in the United States. However, when gender distribution was compared by decade of life, the northwest group had the largest cluster of males in the fourth decade and of females in the second decade. The greatest frequency in both genders occurred in the third decade. There were 18 of 258 (6.9%) male patients with OKC under age 10 in the northwest group and nearly 80% of the patients were Caucasian. The race factor is rarely described in other reports. Gorlin's syndrome was present in 5% of the patients, with a higher distribution in the first and second decades. In conclusion, this is the first report of OKC cases from the Pacific Northwest region of the United States of America.

Adolescent↗

Progression of head and neck cancer in an in vitro model.

OBJECTIVE: To identify alterations in angiogenesis and cell cycle regulation as preneoplastic cells progress to cancer in an in vitro model of head and neck tumor progression. METHODS: Immortal human gingival keratinocyte (IHGK) cells (preneoplastic) were derived from normal oral keratinocytes and were immortalized with human papillomavirus 16. Transformation of IHGK cells with a carcinogen (NNK, 4-[methylnitrosamino]-1-[3-pyridyl]-1-butanone) gave rise to IHGKN cells. We determined the growth rates, cell cycle phase, expression of cell cycle regulators, and expression of vascular endothelial growth factor along with the organotypic features of these cells and compared them with characteristics of head and neck cancer cells. RESULTS: IHGK and IHGKN cells grown in raft culture were morphologically similar to severe dysplasia and carcinoma, respectively. The proportion of cells in G(0)/G(1) was similar between IHGK and IHGKN. However, the proportion of IHGK cells was 35% greater in S phase as compared with the IHGKN cells, while a greater percentage (40%) of IHGKN cells were in G(2)/M. The expression of the other cell cycle regulators tested was unchanged. IHGK cells secreted less vascular endothelial growth factor on day 1 when compared with IHGKN (50.6 vs 245.6 pg/mL), along with a lower overall production rate (79% vs 133%). CONCLUSIONS: Transformation of IHGK cells resulted in the activation of vascular endothelial growth factor associated with angiogenesis. Inactivation of the G(1) cell cycle regulation occurred during immortalization and before transformation, and was sustained after carcinogen exposure. These alterations correspond to changes observed in patients with head and neck squamous cell carcinoma. This model can be useful in testing novel therapeutic and preventive strategies.

Blotting, Western↗

Response of human oral epithelial cells to oxidative damage and the effect of vitamin E.

Smoking and periodontal inflammation are various conditions with the potential to induce oxidative stress and thus DNA damage in the oral cavity. In cellular defense systems, vitamin E is considered the most powerful lipid-soluble antioxidant. To investigate whether oxygen-free radicals alter normal progression of the cell cycle and whether vitamin E prevents this damage, we exposed cultured normal human oral epithelial cells to hydrogen peroxide (H2O2) in the presence or absence of vitamin E. Two primary cell lines were analyzed for the presence of hydroxyl radical, cell cycle distribution and morphology. Each cell line received five treatments: control, ethanol only, vitamin E only, H2O2 only or vitamin E followed by H2O2. Degradation of hydroxyl radicals was detected by electron paramagnetic resonance analysis, cell cycle by flow cytometry and morphology by organotypic technique. Hydroxyl radicals were generated in H2O2-treated cells at an initial concentration, which decreased over a period of time. Cell cycle analysis showed that H2O2-treated cells differed from normal cells in that the percentage of cells in the G1 phase decreased markedly (34.3 vs. 61.2% in control) and the S phase increased (35.5 vs. 15.6% in control). Organotypic cultures treated with H2O2 demonstrated nuclear hyperchromatism, loss of maturation and prominent nucleoli, features consistent with premalignant epithelial transformation. In conclusion, our data suggest that H2O2 produced hydroxyl radicals and altered the cell cycle. Also, vitamin E may have the potential to reduce oxidative damage caused by hydroxyl radicals.

Epithelial Cells↗

5-Fluorouracil-induced apoptosis in cultured oral cancer cells.

Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC.

Antimetabolites, Antineoplastic↗

Effect of brief exposure to mitomycin C on cultured human nasal mucosa fibroblasts.

PURPOSE: To observe the effect of mitomycin C (MMC) on cultured human nasal mucosa fibroblasts. METHODS: Cultured human nasal mucosa fibroblasts were exposed to MMC (0.1-0.4 mg/ml) for 1 to 5 minutes. The viability of the fibroblasts was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay; DNA fragmentation (apoptosis) by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL); apoptotic percentage by flow cytometry; and morphology by light microscopy. RESULTS: A portion of the fibroblasts survived the mitomycin treatment and showed evidence of regrowth within 2 to 3 days. These cells reached confluence in 5 to 7 days. The inhibition rates by MTT assay of 0.4 mg/ml MMC for 5-minute exposures was 31.3%. Dose-response effect was noted with the lower concentrations and shorter exposure times where the inhibition rates were lower (but not significantly so). DNA fragmentation was observed in fibroblasts 24 hours after MMC exposure (0.4 mg/ml) for 5 minutes compared with normal control. The apoptotic rate of fibroblasts treated by 0.4 mg/ml MMC was significantly higher than the control (p < 0.05). CONCLUSIONS: Short MMC exposure times have a variable cytotoxic effect and inhibit proliferation of cultured nasal mucosa fibroblasts. MMC also can induce apoptosis with a 5-minute exposure time. Therefore, it is possible that MMC has a complex effect in dacryocystorhinostomy.

Alkylating Agents↗

Case challenge. Pathological condition of a newborn child.

A new born female presented at birth with a paranasal mass which appeared to be hemangiomatous. An ultrasound and aspiration biopsy of the lesion performed at the time of initial exam were not diagnostic. Due to the rapidly increasing size of the mass, the patient was referred for further evaluation to the Department of Oral and Maxillofacial Surgery at a major northwestern children's hospital. The clinical work up included a bone scan, chest and abdominal CT, and soft tissue biopsy.

Biopsy, Needle↗

The effect of mitomycin C after long-term storage on human Tenon's fibroblast proliferation.

PURPOSE: To investigate the effect of mitomycin C (MMC) after long-term storage on proliferation of human Tenon's fibroblasts in vitro. METHODS: Human Tenon's fibroblasts in tissue culture were exposed for 5 minutes to MMC (0.4 mg/mL) that was either freshly prepared or had been stored for as long as 18 months at either 4 degrees C or -20 degrees C. The MTT colorimetric assay was used to determine the inhibition of proliferation as measured indirectly by mitochondrial activity. RESULTS: The inhibition rate was 88% using fresh MMC, and declined to a mean of 73% when using MMC that had been stored for as long as 18 months at 4 degrees C; this decrease was not statistically significant. The mean inhibition for MMC stored at -20 degrees C was 68%, and this was significantly less than inhibition with fresh MMC. Inhibition did not vary significantly with MMC after different storage times. CONCLUSION: Mitomycin C continues to have strong in vitro antiproliferative effects when stored for as long as 18 months at 4 degrees C or -20 degrees C. A significant decline in potency compared with fresh MMC occurs when MMC is stored at -20 degrees C.

Cell Division↗

Reconstituted human oral and esophageal mucosa in culture.

We have successfully established monolayer and organotypic culture techniques for growing human oral and esophageal epithelial cells. Cells in monolayer culture were grown in serum-free medium, modified from techniques previously reported by our group. The organotypic cultures were grown in a defined medium supplemented with 10% fetal calf serum. Oral and esophageal cells were maintained in keratinocyte basal medium with pituitary extract and other supplements, and 0.05 mM calcium for 7-9 and 9-11 passages, respectively. Both cell types had similar morphology by phase contrast microscopy. When confluent, the cells were predominantly small, basaloid, and uniform and interspersed with larger, differentiated cells. By immunohistochemistry, both cell types in monolayer were positive to AE1, AE3, and 34BE12 antibodies to keratins of stratified epithelia. Oral epithelial cells in monolayer also were positive to 35BH11, representative of simple epithelial keratins, while esophageal cells were not. The esophageal cells were focally positive to K13, while the oral cells were negative. Both were negative for K19. When comparing monolayer to organotypic cultures and to in vivo specimens, there was a significant difference in the expression of keratins. Using organotypic cultures, AE1, AE3, and 34BE12 were strongly positive in both oral and esophageal cells, similar to in vivo tissues. In contrast to monolayers, both were also focally positive for K19. Esophageal cells were strongly positive for K13, while the oral cells were mildly but uniformly positive. Both were negative for keratins of simple epithelia. These two cell culture techniques offer unique opportunities to study the pathobiology, including carcinogenesis, of stable cell systems from the oral and esophageal epithelia.

Cells, Cultured↗

Culture of human main pancreatic duct epithelial cells.

Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated culture inserts suspended above a human fibroblast feeder layer. After primary culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-free medium continued to develop into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These cells were also positive for cytokeratin 19, 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry. Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-D-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passaged using a feeder layer system, with serum-free medium, and in organotypic cultures.

Cell Culture Techniques↗

Odontogenic ghost cell carcinoma: a case report with immunohistochemical and ultrastructural characterization.

The odontogenic ghost cell tumor (OGCT) is the rare, neoplastic variant of the calcifying odontogenic cyst (COC). To date, there have been only four reports of malignant OGCTs, in five patients. We report an additional case of a malignant odontogenic ghost cell tumor that involved the right maxilla of a 20-year-old man. Our report includes its immunocytochemical and ultrastructural characterization.

Adult↗

Smokeless tobacco-exposed oral keratinocytes increase macromolecular efflux from the in situ oral mucosa.

The purpose of this study was to determine whether supernatants of cultured human oral keratinocytes (HOK) exposed to an aqueous extract of smokeless tobacco (STE) increase macromolecular efflux from the oral mucosa in vivo and, if so, whether bradykinin mediates in part this response. Subconfluent monolayers of HOK were incubated with STE or media, and supernatants were collected 24, 48, and 72 h thereafter. Using intravital microscopy, we found that suffusion of supernatants of STE- but not media-exposed HOK elicited significant concentration- and time-dependent increases in efflux of fluorescein isothiocyanate-labeled dextran (mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). These effects were significantly attenuated by HOE-140 and NPC-17647 but not by des-Arg9, [Leu8]-bradykinin. Proteolytic activity was increased in supernatants of STE- but not media-exposed HOK. However, a mixture of leupeptin, Bestatin, and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid had no significant effects on HOK supernatant-induced responses. Collectively, these data suggest that oral keratinocytes modulate smokeless tobacco-induced increase in macromolecular efflux from the in situ oral mucosa in part by elaborating proteases that may account for local bradykinin production.

Animals↗

Head and neck osteosarcoma at the University of Washington.

BACKGROUND: Head and neck osteosarcoma is a comparatively rare and aggressive malignancy. Our goal was to examine the experience of head and neck osteosarcoma patients seen over a 15-year period at the University of Washington Medical Center and compare this with the published experience of other centers in terms of demographics, histology, treatment, and survival rate. METHODS: We reviewed surgical pathology slides and clinical treatment records of 13 patients who were treated at the University of Washington Medical Center between 1981 and 1996. A total of 17 cases from 13 patients (13 primary tumors and 4 recurrences) were studied. RESULTS: There was a slight male predominance, with a male:female ratio of 1.6:1, and median age at diagnosis of 40.9 years (range 22 to 75 years), both slightly higher than has been generally reported. Three of 13 patients had recognized risk factors for the development of osteosarcoma: 2 with a history of prior radiotherapy and 1 with Paget's disease. All surgical pathology specimens were examined independently by two pathologists for histologic grading and typing. At initial presentation, 9/13 (69%) cases had conventional (osteoblastic) histology; 2/13 (15%) were fibroblastic, 1 chondroblastic (8%) and 1 parosteal (8%). Eight of 13 (62%) cases were high grade at initial presentation. Four of 13 (30%) of the primary tumors were low grade 2, of which did not recur over a median follow-up period of 24 months. The other 2 low-grade tumors later recurred locally, as high-grade osteosarcomas, after disease-free intervals of 1 year and 14 years, respectively. One patient had an intermediate-grade tumor which has not recurred as of last follow-up. Combined-modality treatment, including surgery with or without radiotherapy and/or chemotherapy, was given depending on the histologic grade, surgical margins, and recurrence. Some patients with low-grade tumors had surgery only. There were 5 local recurrences, 1 of these following a disease-free interval of 14 years. One patient had 3 separate recurrences at the same site. Ten of 13 (77%) are alive and disease-free. Of the 3 deaths, 1 was related to radiation-induced brain necrosis, without evidence of recurrent tumor. The project 5-year overall survival in this series is 72%, with a mean follow-up of 58 months (median, 36 months). Of those receiving neoadjuvant chemotherapy, 6/7 have survived to the present. CONCLUSION: Given the limitations of a small patient population, our data suggest that neoadjuvant chemotherapy may provide benefit in terms of survival. Longer follow-up will be necessary to support this conclusion. Our data also show that our population has a higher-than-average age of onset, low presence of risk factors, and better survival rate in comparison with the published series from other institutions.

Adult↗

The effect of N-methyl-N'-nitro-N-nitrosoguanidine on cultured dog gallbladder epithelial cells.

Normal dog gallbladder epithelial cells in long-term culture were used as a model to study the morphologic, genetic, and secretory processes associated with the progression to cancer formation. Dog gallbladder epithelial cells cultured on collagen-coated plates grew into polarized monolayers, could be passaged repeatedly, and showed the typical morphological profile of well-differentiated columnar epithelial cells. After cells were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 10(-5) mol/L for 48 hours, the treated cells grew on plastic and could be cloned. Flow cytometry revealed emergence of an aneuploid cell subpopulation. In organotypic culture, treated cells showed a pseudostratified appearance, with cellular and nuclear pleomorphism. Large and hyperchromatic nuclei were present as well as increased mitotic rate. The proteins of MNNG-treated dog gallbladder epithelial cells showed increased phosphorylation of tyrosine residues. Treated cells showed a decrease in mucin secretion in response to prostaglandin E2, manifesting an altered pattern of mucin secretion. Transforming growth factor-beta failed to inhibit cell proliferation in the MNNG-treated cells compared with the prominent inhibition in normal cells. Together, the data reflected changes representing preliminary steps on the pathway to develop cancer cells. Our results indicate that carcinogenic chemicals can cause measurable chromosomal and cellular modifications to normal biliary epithelial cells in vitro. This model may be useful in understanding the sequential steps in carcinogenesis and affords an opportunity to study chromosomal damage, cytokinetics, changes in molecular genetic markers, and expression, as well as cell biological function during cellular transformation.

Animals↗

Spontaneous exfoliation of teeth following severe elemental mercury poisoning: case report and histological investigation for mechanism.

BACKGROUND: Although the spontaneous exfoliation of teeth and breakdown of oral tissues from severe mercury intoxication have been noted for over a century, there are no published reports investigating the mechanisms of these phenomena. Severe mercury poisoning is rare in modern times, but it does occur. We present a case report and a histopathologic investigation into the mechanism of the associated tooth loss. METHODS: An exfoliated tooth and periodontal and gingival tissues were obtained from a 15-month-old patient who had been severely intoxicated with elemental mercury over a period of months and hospitalized for severe neurologic and renal effects. The tissues were examined both by routine hematoxylin and eosin stain and by autometallography specific for mercury. For comparison, control tissue from an age-matched subject was examined with the autometallography technique. RESULTS: Under light microscopy, the gingival tissue showed evidence of moderate to severe acute and chronic inflammation. The tooth pulp tissue showed evidence of moderate vascular dilatation and congestion, and it was infiltrated by many neutrophils. The autometallographic sections showed intense accumulations of mercury in the soft tissues of the mercury-exposed subject, but not in the tissues of the control subject. The deposits were primarily found in fibroblasts, which are essential to maintaining the integrity of the oral tissues. CONCLUSIONS: Histopathologic and autometallographic examination of the affected tissue indicates that the primary mechanism of the spontaneous sloughing of tissue and loss of teeth may be the cytotoxic effects of the accumulation of mercury in fibroblasts. Studies of additional cases would be valuable to confirm this hypothesis.

Case-Control Studies↗

Coating of titanium alloy with soluble laminin-5 promotes cell attachment and hemidesmosome assembly in gingival epithelial cells: potential application to dental implants.

The formation of a biological seal around the transmucosal portion of dental implants may be crucial for the long-term success of these therapies. Data to date suggest that the gingival epithelium attaches to dental implants through the formation of hemidesmosomes. Biochemical and genetic data indicate that the laminin isoform, laminin-5, a component of basement membranes, plays a crucial role in the assembly and maintenance of hemidesmosomes. We report the use of soluble laminin-5 as a biological coating of titanium-alloy to promote cell attachment of the gingival epithelial cell line, IHGK. Monoclonal antibodies reactive with laminin-5 depleted the coating solution of all cell attachment activity and blocked cell attachment to laminin-5-coated disks. Immunodepletion with antibodies to fibronectin had no effect. Finally, we demonstrate that IHGK cells assembled hemidesmosomes within 24 h of attachment to laminin-5-coated titanium alloy but not to the titanium alloy alone. These results suggest that soluble laminin-5 may have clinical applications as a dental implant coating to promote the formation of a biological seal.

Alloys↗

HPV immortalization of human oral epithelial cells: a model for carcinogenesis.

Human papillomavirus (HPV) has been implicated in the etiology of oral and cervical cancers. Normal oral epithelial cells at passage two were infected with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. The G418-selected cells that were immortalized and express HPV 16 E6/E7 have been in culture for over 4 years and 350 passages. In contrast, the normal oral epithelial cells did not survive the culture environment beyond 7 to 9 passages. Fifteen clones were selected from the pooled population. By Northern blot analysis all clones demonstrated the presence of E6solidusE7 genes. Keratin expression of both normal and immortalized oral epithelial cells was studied in organotypic culture. Both cell types were positive with antibodies AE1, AE3, and 34BE12. Both were focally positive with AE8, which stains for keratin 13 (specific for oral and esophageal epithelial cells). The normal control cells were focally positive for filaggrin, while the HPV 16-immortalized cells (IHGK cells-immortalized human gingival keratinocytes) were negative. The IHGK cells were strongly positive with KS19.1, which stains the embryonal keratin K19, an indicator of premalignant or malignant changes, while the normal control cells were only lightly and focally positive. In conclusion, we present an oral epithelial cell line successfully immortalized with HPV E6/E7 which will facilitate further research on the involvement of HPV in oral carcinogenesis.

Cell Transformation, Viral↗