Exosomes: Nomenclature, Isolation, and Biological Roles in Liver Diseases

The biogenesis and biological roles of extracellular vesicles (EVs) in the progression of liver diseases have attracted considerable attention in recent years. EVs are membrane-bound nanosized vesicles found in different types of body fluids and contain various bioactive materials, including proteins, lipids, nucleic acids, and mitochondrial DNA. Based on their origin and biogenesis, EVs can be classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are the smallest EVs (30-150 nm in diameter), which play a significant role in cell-to-cell communication and epigenetic regulation. Moreover, exosomal content analysis can reveal the functional state of the parental cell. Therefore, exosomes can be applied to various purposes, including disease diagnosis and treatment, drug delivery, cell-free vaccines, and regenerative medicine. However, exosome-related research faces two major limitations: isolation of exosomes with high yield and purity and distinction of exosomes from other EVs (especially microvesicles). No standardized exosome isolation method has been established to date; however, various exosome isolation strategies have been proposed to investigate their biological roles. Exosome-mediated intercellular communications are known to be involved in alcoholic liver disease and nonalcoholic fatty liver disease development. Damaged hepatocytes or nonparenchymal cells release large numbers of exosomes that promote the progression of inflammation and fibrogenesis through interactions with neighboring cells. Exosomes are expected to provide insight on the progression of liver disease. Here, we review the biogenesis of exosomes, exosome isolation techniques, and biological roles of exosomes in alcoholic liver disease and nonalcoholic fatty liver disease.

Real-world Treatment Pattern and Outcomes of Hypercalcemia among Solid Tumor Patients

BACKGROUND: Hypercalcemia is an important metabolic emergency condition in cancer patients. Bisphosphonate is the treatment of choice for hypercalcemia, whereas calcitonin and hydration with furosemide are recommended for acute supportive therapy. However, data regarding real-world treatment patterns and outcomes of pharmacological treatments are limited. Therefore, we aimed to investigate the treatment patterns and clinical outcomes of hypercalcemia treatment in solid tumor patients. METHODS: Electronic medical records of 123 adults with solid cancers and albumin-corrected calcium levels >10.5 mg/dL or ionized calcium levels >1.35 mmol/L were reviewed. We retrospectively analyzed the pharmacological treatment and recovery rate according to the severity of hypercalcemia. RESULTS: A total of 177 cases were identified, of which 49 were not treated and 30 were treated with hydration only. In moderate-to-severe cases, 86.5% received pharmacological treatment. Thirty-four cases (19.2%) were treated with bisphosphonate alone and 58 cases (32.8%) were treated with bisphosphonate and calcitonin. In mild hypercalcemia cases, the recovery rate was higher for those receiving hydration only or pharmacological treatment (79.7%) than for those receiving no treatment (61.4%, p = 0.041). Most moderate-to-severe cases were treated with medication and of those treated, 56.3% recovered. The recovery rate was lower in those treated with bisphosphonate alone (38.2%) than in those who underwent calcitonin combination treatment (73.7%, p = 0.001). CONCLUSIONS: Bisphosphonate combined with calcitonin was found to be more effective than bisphosphonate alone for the treatment of moderate-to-severe hypercalcemia. Considering the current shortage of calcitonin, further efforts are required to ensure its stable supply.

Flurbiprofen toxicity in 2 dogs / 대한수의학회지

Two dogs were presented with melena, vomiting and depression after accidental swallowing of candy form of Strepsils (flurbiprofen), which is one of non-steroidal anti-inflammatory drugs used in human medicine for controlling a sore throat. These dogs had common signs of anemia induced by gastrointestinal ulceration and hemorrhage with azotemia and leukocytosis. The dogs were treated with blood transfusion, fluid therapy, proton-pump inhibitor, antiemetics, mucus protectant and antibiotic. Although most of clinical signs of two dogs were resolved, azotemic problem with evidence of renal injury have remained.

Clinical use of a ceramide-based moisturizer for treating dogs with atopic dermatitis

In humans, skin barrier dysfunction is thought to be responsible for enhanced penetration of allergens. Similar to conditions seen in humans, canine atopic dermatitis (CAD) is characterized by derangement of corneocytes and disorganization of intercellular lipids in the stratum corenum (SC) with decreased ceramide levels. This study was designed to evaluate the effects of a moisturizer containing ceramide on dogs with CAD. Dogs (n = 20, 3~8 years old) with mild to moderate clinical signs were recruited and applied a moisturizer containing ceramide for 4 weeks. Transepidermal water loss (TEWL), skin hydration, pruritus index for canine atopic dermatitis (PICAD) scores, and canine atopic dermatitis extent and severity index (CADESI) scores of all dogs were evaluated. Skin samples from five dogs were also examined with transmission electron microscopy (TEM) using ruthenium tetroxide. TEWL, PICAD, and CADESI values decreased (p < 0.05) and skin hydration increased dramatically over time (p < 0.05). Electron micrographs showed that the skin barrier of all five dogs was partially restored (p < 0.05). In conclusion, these results demonstrated that moisturizer containing ceramide was effective for treating skin barrier dysfunction and CAD symptoms.

Evaluation of the effect of a 0.0584% hydrocortisone aceponate spray on clinical signs and skin barrier function in dogs with atopic dermatitis

The purpose of this study was to evaluate the effects of a topical spray containing 0.0584% hydrocortisone aceponate (HCA) on canine atopic dermatitis (CAD) and to evaluate the skin barrier function during the treatment of CAD. Twenty-one dogs that fulfilled the diagnostic criteria for CAD were included in this study. The HCA spray was applied once a day to the lesions of all dogs for 7 or 14 days. Clinical assessment was performed before (day 0) and after treatment (day 14), and clinical responses were correlated with changes in skin barrier function. CAD severity significantly decreased after 14 days of HCA treatment based on the lesion scores (p < 0.0001), which were determined using the CAD extent and severity index (CADESI-03) and pruritus scores (p < 0.0001) calculated using a pruritus visual analog scale. Transepidermal water loss, a biomarker of skin barrier function, was significantly reduced compared to baseline (day 0) measurements (p = 0.0011). HCA spray was shown to be effective for significantly improving the condition of dogs suffering from CAD. This treatment also significantly improved cutaneous hydration and skin barrier function in the animals.

Detection of Epstein-Barr virus in the inflammatory and neoplastic uterine cervical lesions / 대한세포병리학회지

The prevalence of Epstein-Barr virus(EBV) in the uterine cervix was investigated to define the possible etiologic role in cervical carcinogenesis. The viral genotyping and LMP-1 30bp deletion were also studied. The materials included 169 uterine cervical swabs(152 within normal limits, 12 atypical squamous cells of uncertain significance, 3 low grade intraepithelial lesions, and 2 high grade squamous intraepithelial lesion) and 104 uterine cervical tissues obtained from hysterectomy specimens(32 carcinoma in situ, 9 microinvasive squamous cell carcinomas, 37 invasive squamous cell carcinomas, 7 adenocarcinomas, 7 adenosquamous carcinomas, and 12 cervicitis). EBV detected by PCR for EBNA-1 was positive in 52(56.5%) of 92 invasive and noninvasive cervical carcinomas, and 80(48.8%) of 164 inflammatory or normal cervices. The viruses detected in carcinomas were all type A, and LMP-1 30bp deletion form was more frequent in premalignant and malignant cervical lesions than in nonneoplastic cervices. From the above results, it may be concluded that EBV is one of common viruses detected in uterine cervix of Korean women, and type A virus and LMP-1 30bp deletion form may have a role in cervical carcinogenesis.

Expression of Cancer-Related Genes in Epstein Barr Virus-Infected Burkitt's Lymphoma Cell Line Treated with Mitomycin C

BACKGROUND: Infection of Epstein Barr virus (EBV) into B cells drives the infected cells into the cell cycle and frequently results in lymphoblastoid cells. Mitomycin C inhibits DNA synthesis of epithelial cells as well as lymphoid cells by cross-linking with DNA. Many of the cancer cells have various pathways for escaping the responsiveness to the negative growth-regulatory effects of mitomycin C and gaining the immortalized property. The auther performed a cell culture of an EBV infected Jijoye lymphoma cell line, and compared the cell cycle and cancer related genes between the mitomycin treated- and non-treated group. METHODS: DNA and RNA were extracted from the Jijoye cells; and EBV nuclear antigen (EBNA)-1, 2 and latent membrane protein (LMP) of EBV and p53 and p21 mRNA analyse was performed. RESULTS: Mitomycin C blocked G2/M phase, however, mitomycin did not affect the expression of EBNA-1, 2 and LMP. Mitomycin C also increased the p21 mRNA expression without p53 mRNA increase. CONCLUSIONS: Mitomycin C induces B cell apoptosis by blocking the G2/M phase and by increasing p21 mRNA independent to p53, which reveals the presence of an alternative pathway of p21 induction by mitomycin C in EBV positive lymphoma cells

The detection of circulating breast cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction

Some circulating cancer cells in the blood play a central role in the metastatic process and may have a major influence on patient progress. Their numbers can be very small and techniques for their detection need to be both sensitive and specific. Polymerase chain reaction (PCR) has been successfully used to detect small numbers of tumor cells in cancer. We used a reverse transcriptase-polymerase chain reaction (RT-PCR) to detect circulating breast cancer cells in venous blood samples before operations and assessed cytokeratin-19 (CK-19) and cytokeratin-20 (CK-20) as target mRNA markers in the blood of healthy donors (n=6) and breast cancer patients (n=30) with American Joint Committee on Cancer stages 0 to IIIa. CK-19 mRNA was expressed in all blood samples of healthy donors and patients. But CK-20 was the only mRNA marker not detected in the blood from healthy donors. Seven of 30 (23%) venous blood isolates of breast cancer patients yielded a CK-20 mRNA with positive results. There was no correlating CK-20 mRNA expression with stage and axillary lymph node status. In conclusion, CK-19 showed no diagnostic value as a mRNA marker in the detection of circulating cancer cells by RT-PCR assay because this was expressed in the blood of healthy donors. CK-20 mRNA was an useful marker to detect circulating cancer cells in breast cancers.

Histopathologic Findings, and p53 and K-ras Mutational Analysis in Biopsy Specimens Using Fluorescence Bronchoscopy

A fluorescence bronchoscope system has been developed for detecting early lung cancer including dysplasia and carcinoma in situ. To determine the histologic findings and genetic alterations of the lung tissues, which were biopsied by the fluorescence bronchoscope, we analyzed 104 specimens from 62 heavy smokers for their histopathology, cell proliferation index, and genetic mutations of p53 and K-ras. We used immunohistochemistry for MIB-1 and p53, and PCR-SSCP and direct DNA sequencing for p53 and K-ras. The histology was variable from reactive conditions to invasive cancers, and consisted of basal cell hyperplasia (26.9%), dysplasia (4.8%), carcinoma in situ (1.9%), squamous cell carcinoma (7.7%), adenocarcinoma (4.8%), and small cell carcinoma (10.6%). The cellular proliferation index of the lesions increased as their aggressiveness increased. p53 and K-ras mutations were detected in 33.7% and 14.4% of all tissues, respectively. In dysplasia, p53 and K-ras mutations were observed in 3 of 5 and in 2 of 5 tissues, respectively. However, these genetic alterations were not found in carcinoma in situ. Interestingly, 28.6% of basal cell hyperplasia showed p53 mutations. In conclusion, these data suggest that the biopsy specimens using fluorescence bronchoscopy show variable histologic findings, ranging from reactive conditions to invasive cancers. In addition, some of the dysplastic lesions are related to p53 and K-ras mutations, although these genetic alterations are also seen in basal cell hyperplasia.

Role of p53 gene mutation in tumor aggressiveness of intracranial meningiomas

The mutations that occur in the p53 tumor suppressor gene have been studied in various human malignant tumors. However, little is known about this gene in meningiomas. To investigate the relationship and frequency of p53 gene mutations, the p53 polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and immunohistochemical study were performed on the 41 intracranial meningiomas (21 benign, 11 atypical, and 9 malignant). The higher the p53 protein expression rate, the poorer the histologic grade (9.5%, 72.7%, and 88.9% in benign, atypical and malignant meningioma, respectively) (p=0.000). The p53 protein expression rate was higher in recurrent meningioma (71.4%) than in nonrecurrent meningioma (10.5%) (p=0.002). PCR-SSCP method was performed in positive p53 protein immunoreactivity cases. p53 gene mutation rate was higher in the atypical (62.5%) and malignant (25%) meningiomas than in the benign meningioma (0%) (p=0.232). Also, the rate was higher in recurrent menigioma (20%) than in nonrecurrent meningioma (0%) (o=0.495). Among five to eight exons of the p53 gene, the mutation was observed on exon 7 more frequently. In conclusion, p53 immunoreactivity and p53 gene mutation are closely correlated with histologic grade and histologic atypia of intracranial meningiomas. p53 gene mutation would be considered as a useful marker to detect the progression of intracranial meningiomas.

p53 Gene Mutation in Epithelial Ovarian Cancer / 대한산부인과학회잡지

OBJECTIVE: Although ovarian cancer is the leading cause of death among all cancers of the female reproductive tract, the genetic alterations involved in ovarian cancer remains largely unknown. Recently, mutations of the p53 gene have been documented in many types of human cancer including ovarian cancer. METHODS: In tbe present study, p53 gene mutation was examined in DNA samples extracted from paraffin embedded surgical specimens of ovarian cancer. Furthermore, clinicopathological parameters were examined in relation to p53 gene mutation in order to understand the role of p53 mutation in the development of ovarian cancer. Using the polymerase chain reaction(PCR) and single strand conformational polymarphism(PCR-SSCP), p53 gene mutation was examined and the mutations were confirmed by DNA scquencing in 17 cases of ovarian cancer. RESULTS: Abnormal bands indicating mutation were detected in 2/17(11.8%). DNA sequencing confirmed in 2 mutations and revealed C to T and A to T nucleotide chmges. In clinicopathological parameters, FIGO stage, grade, and recunence were not correlated with the p53 gene mutation. However, the recurrence rate was higher in patients with mutant p53 compared with those with wild type p53(50.0% vs 13.3%), altbough this is not statisticaUy significant. CONCLUSION: In conclusion, p53 gene mutation shows no correlation with stage, grade and recurrence, and p53 gene mutation does not appear to be a marker that predicts the biological behavior or the outmme of the disease. This study suggested useful data to elucidate the mechanism of chemotherapy-resistant ovarian cancer and further p53 expression assay would be mandatory for p53 nonfunctioning ovarian cancas.

A Comparative Study of the Detection of the p53 Abnormality in Breast Cancer by Using PCR-SSCP and Antibody Staining

BACKGROUND: Mutations eliminating or altering the p53 protein function are the single most common genetic alteration in nearly all types of human cancers. The project of the p53 gene is hypothesized to maintain genomic stability by blocking cell replication or by initiating apoptosis after DNA damage. Many p53 mutations alter the conformation of the protein, which results in abnormal overexpression. METHODS: This study investigated the correlation between p53 mutations detected at the DNA level and the p53 protein expression determined by immunohistochemical staining. Abnormalities of the p53 gene and protein in 30 primary paraffin embedded breast cancer tissues were examined. RESULTS: Mutations in p53 exons 5-8 were identified in 9 of the 30 cases (30%) by using a polymerase chain-reaction single stranded conformational polymorphism (PCR-SSCP) analysis. Overexpression of the p53 protein was detected in 11 of the 30 cases (37%) by using mouse monoclonal p53 antibody (Zymed Essence Co.) Positive immunohistochemical staining without mutations was detected by PCR-SSCP analysis in 4 cases, but a mutation with negative immunohistochemical staining was detected by PCR-SSCP analysis in only one case. p53 abnormality was not associated with TNM stages. The sensitivity between these methods was 73%. CONCLUSIONS: Positive immunohistochemical staining using p53 monoclonal antibody could detect p53 protein expression, but this result did not correlate completely with p53 mutation in exon 5-8.

Detection of Human Papillomavirus(HPV) in Sinonasal Inverted Papillomas Using Polymerase Chain Reaction(PCR) / 대한이비인후과학회지

The human papillomavirus(HPV) is a subfamily of the Papovaviridae family as a double stranded DNA virus, and HPV is the etiological agent of squamous cell papillomas in different anatomic regions including the skin and the mucous membranes of oral cavity, esophagus, respiratory and anogenital tracts. Inverted papilloma of the nasal cavity and paranasal sinuses is uncommon benign lesion, in which there is a inversion of the neoplastic epithelium into the underlying stroma. The local aggressiveness, high rate of recurrence, associated malignancy, and tendency of multicentricity have led to the advocation of radical removal of the tumor. The cause of sinonasal inverted papilloma remains unknown. However, the involved etiologic factors are thought to be smoking, allergy, environmental factors, HPV, and chronic infections. The purpose of this study was to detect HPV in sinonasal inverted papilloma, to examine the relationship between cellular dysplasia and recurrence of inverted papilloma, to examine the relationship between HPV and recurrence of inverted papilloma in forty two sinonasal inverted papillomas(inverted papilloma without dysplasia 30 cases, inverted papilloma with dysplasia 6 cases, inverted papilloma associated squamous cell carcinoma 5 cases, inverted papilloma transformed squamous cell carcinoma 1 case). For these purposes, paraffin-embedded tissues were subjected to polymerase chain reaction using type-specific primers pairs. Following results were obtained: 1) The HPV was detected in 5(12%) out of 42 cases of inverted papilloma, one contained human papillomvirus 6, two contained human papillomavirus 11, and two contained human papillomavirus 16. 2) The recurrence of inverted papilloma occurred in 1(16%) out of 6 cases exhibited dysplasia, in 3(10%) out of 30 cases not exhibited dysplasia. 3) The recurrence of inverted papilloma occurred in 2(66%) out of 3 cases positive for HPV, in 2(6%) out of 33 cases negative for HPV. In conclusion HPV was thought to be the etiological factor of sinonasal inverted papilloma. Also there was a relationship between HPV and recurrence of inverted papilloma. Further work is in progress to determine the possible mechanisms by which HPV induces oncogenesis in inverted papilloma.

TGF-beta-1 Expression and p53 Mutation in Non-small Cell Carcinomas of the Lung / 대한암학회지

PURPOSE: TGF-beta-1 is actually a major growth inhibitor for most cell types. We assumed that the loss of TGF-beta-1 would be occurred during carcinogenesis of the lung. Also, the mutation and expression of p53 have been known to be major moleclar change of non-small cell carcinoma of the lung. So, the relationship between the mutation of p53 and the expression of TGF-beta-1 in the non-small cell carcinomas were evaluated. MATERIALS AND METHODS: In 43 non-small cell carcinoma and normal tissue of the lung, their TGF-beta-1 mRNA were measured by RT-PCR and p53 was studied by SSCP and Western blotting assay. RESULTS: p53 mutation rate in non-small cell carcinomas of the lung (48.4%) was much more frequent than the normal control group (14.3%). The expression rate of TGF-beta-1 in lung carcinomas, especially squamous cell carcinoma (71.4%), was much higher than the normal control group (42.9%). p53 mutation and TGF-beta-1 mRNA in the lung carcinomas were not strongly correlated. CONCLUSION: It suggests that high expression rate of TGF-beta-1 and p53 mutation are associated with carcinogenesis of non-small cell carcinoma of the lung. High expression rate of TGF-beta-1 in the lung carcinomas can be partly explained by the fact that TGF-beta-1 have capacity to control the production of many components of the extracellular matrix and enhance angiogenesis in favor of tumor growth despite of their inhibitory effects of cell growth. However, additional research is required to determine the exact role of TGF-beta-1 in carcinogenesis of the lung.

Detection and Subtyping of Epstein-Barr Virus in Gastrointestinal Adenocarcinomas and Malignant Lymphomas

Epstein-Barr virus (EBV) has been linked to a spectrum of neoplastic conditions, including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, lymphoepithelioma-like carcinomas and malignant lymphomas in immunocompromised state. To determine the prevalence and the subtype of EBV in gatrointestinal malignancies, fifty cases of adenocarcinomas and seventeen cases of malignant lymphomas were analyzed by EBERs in situ hybridization and polymerase chain reaction using primers for EBNA-1, EBNA-2A and EBNA-2B, on the paraffin sections. In addition, immunohistochemical stain for p53 protein was performed to investigate the potential role of EBV infection on tumor suppressor gene, p53, during tumorigenesis. EBER was detected in 6 of 26 gastric adenocarcinomas, 2 of 24 colon adenocarcinomas, and 8 of 17 malignant lymphomas. EBER was more prevalent in malignant lymphoma arising in the intestine (6/6) than in the stomach (2/11), and was detected in both B and T cell phenotypes. EBNA-1 was positive in 11 of 16 EBER positive cases and the subtyping was possible in 8; both type 1 and 2 were detected in gastric cancers, whereas only type 2 was found in intestinal neoplasms. In adenocarcinomas the high rate of p53 protein overexpression was found in both EBER positive (8/8) and negative cases (32/42), whereas the positive rate was higher in EBER positive cases (7/8) than in EBER negative cases (4/9) of malignant lymphomas. From the results, it can be concluded that EBV infection and the p53 tumor suppressor gene are independently associated in a significant portion of the gastrointestinal malignancies, but the mechanism of action remains to be elucidated.