Ok Jun Lee

The Polymerase Acidic Protein Gene of Influenza A Virus Contributes to Pathogenicity in a Mouse Model

ABSTRACT Adaptation of influenza A viruses to a new host species usually involves the mutation of one or more of the eight viral gene segments, and the molecular basis for host range restriction is still poorly understood. To investigate the molecular changes that occur during adaptation of a low-pathogenic avian influenza virus subtype commonly isolated from migratory birds to a mammalian host, we serially passaged the avirulent wild-bird H5N2 strain A/Aquatic bird/Korea/W81/05 (W81) in the lungs of mice. The resulting mouse-adapted strain (ma81) was highly virulent (50% mouse lethal dose = 2.6 log10 50% tissue culture infective dose) and highly lethal. Nonconserved mutations were observed in six viral genes (those for PB2, PB1, PA, HA, NA, and M). Reverse genetic experiments substituting viral genes and mutations demonstrated that the PA gene was a determinant of the enhanced virulence in mice and that a Thr-to-Iso substitution at position 97 of PA played a key role. In growth kinetics studies, ma81 showed enhanced replication in mammalian but not avian cell lines; the PA97I mutation in strain W81 increased its replicative fitness in mice but not in chickens. The high virulence associated with the PA97I mutation in mice corresponded to considerably enhanced polymerase activity in mammalian cells. Furthermore, this characteristic mutation is not conserved among avian influenza viruses but is prevalent among mouse-adapted strains, indicating a host-dependent mutation. To our knowledge, this is the first study that the isoleucine residue at position 97 in PA plays a key role in enhanced virulence in mice and is implicated in the adaptation of avian influenza viruses to mammalian hosts.

Identification of suitable reference genes for the relative quantification of microRNAs in pleural effusion

Circulating cell-free microRNAs (miRNAs) are potential biomarkers of cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is widely used in miRNA expression studies. The aim of this study was to identify suitable reference genes for RT-qPCR analyses of miRNA expression levels in pleural effusion. The expression levels of candidate reference miRNAs were investigated in 10 benign pleural effusion (BPE) and 10 lung adenocarcinoma-associated malignant pleural effusion (LA-MPE) samples using miRNA microarrays. The expression levels of candidate reference miRNAs, together with those of U6 small nuclear RNA (snRNA), RNU6B, RNU44 and RNU48 small RNAs, in 46 BPE and 45 LA-MPE samples were validated by RT-qPCR, and were analyzed using the NormFinder and BestKeeper algorithms. The impact of different normalization approaches on the detection of differential expression levels of miR-198 in BPE and LA-MPE samples was also assessed. As determined by the miRNA microarray data, five candidate reference miRNAs were identified. Following RT-qPCR validation, U6 snRNA, miR-192, miR-20a, miR-221, miR-222 and miR-16 were evaluated using the NormFinder and BestKeeper software programs. U6 snRNA and miR-192 were identified as single reference genes and the combination of these genes was preferred for the relative quantification of miRNA expression levels in pleural effusion. Normalization of miR-98 expression levels to those of U6 snRNA, miR-192 or a combination of these genes enabled the detection of a significant difference between BPE and LA-MPE samples. Therefore, U6 snRNA and miR-192 are recommended as reference genes for the relative quantification of miRNA expression levels in pleural effusion.

Evaluation of Urine Cytology in Urothelial Carcinoma Patients: A Comparison of CellprepPlus® Liquid-Based Cytology and Conventional Smear

Background Urine cytology is an important test in the screening of urothlelial neoplasms. The conventional smear (CS) method of testing urine samples has a low sensitivity, approximately 50% result accuracy for detecting urothelial carcinomas, while liquid-based cytology (LBC) has much improved diagnostic accuracy, sensitivity, and specificity. The aim of this study was to compare the morphologic features and diagnostic efficacy of CellprepPlus® LBC with those of CS for urine cytology. Methods A total of 713 cases of urine specimens collected from November 2009 to September 2010 were included. All specimens were divided equally for the preparation of CellprepPlus® LBC and CS for each case. Results CellprepPlus® revealed more cellularity, a cleaner background and better cytomorphologic features, but it showed a less intact architectural pattern compared to that of CS. Of the 88 histologically confirmed cases, the diagnostic sensitivity for CellprepPlus® was 50% and higher than the 37.5% for CS. The specificity of both preparations was 100%. Conclusions The CellprepPlus® showed an improved quality of slides and provided better diagnostic accuracy, thus CellprepPlus® could be a first-line screening tool in urinary tract cytology.

Metastatic Renal Cell Carcinoma in a Supraclavicular Lymph Node with No Known Primary: A Case Report

Although metastasis is relatively frequent in cases of renal cell carcinoma (RCC), metastasis in the cervical or supraclavicular lymph node (LN) is relatively rare. Moreover, cases of metastatic RCC with a non-identifiable kidney mass are extremely rare. Here, the authors report a case of metastatic RCC in a supraclavicular LN without a primary kidney lesion. A 69-year-old man presented with a progressively enlarging right supraclavicular mass. Incisional biopsy of the affected supraclavicular LN was performed, and histological examination revealed metastatic RCC. However, no tumor was found in either kidney, despite various examinations. The patient was treated with radiotherapy followed by sunitinib. After three months on sunitinib, a follow-up computed tomography scan revealed that the supraclavicular LN had markedly decreased, and after 20 months, the disease had not progressed. This case suggests that, even when there is no primary kidney lesion, clinicians must consider the possibility of metastatic RCC when evaluating patients with clear cell carcinoma with an unknown primary site.

Early Regulation of Viral Infection Reduces Inflammation and Rescues Mx-Positive Mice from Lethal Avian Influenza Infection

Differing sensitivity of influenza A viruses to antiviral effects of the Myxovirus resistance (Mx) protein implies varying global gene expression profiles in the host. The role of Mx protein during lethal avian influenza (AI) virus infection was examined using Mx1-deficient C57BL/6 (B6-Mx1(-/-)) and congenic Mx1-expressing (B6-Mx1(+/+)) mice infected with a virulent, mouse-adapted avian H5N2 Ab/Korea/ma81/07 (Av/ma81) virus. After infection, B6-Mx1(+/+) mice were completely protected from lethal AI-induced mortality, and exhibited attenuated clinical disease and reduced viral titers and pathology in the lungs, compared with B6-Mx1(-/-) mice. Transcriptional profiling of lung tissues revealed that most of the genes up-regulated after infection are involved in activation of the immune response and host defense. Notably, more abundant and sustained expression of cytokine/chemokine genes was observed up to 3 dpi in B6-Mx1(-/-) mice, and this was associated with excessive induction of cytokines and chemokines. Consequently, massive infiltration of macrophages/monocytes and granulocytes into lung resulted in severe viral pneumonia and potentially contributed to decreased survival of B6-Mx1(-/-) mice. Taken together, our data show that dysregulated gene transcriptional activity corresponded to persistent induction of cytokine/chemokines and recruitment of cytokine-producing cells that promote inflammation in B6-Mx1(-/-) mouse lungs. Thus, we provide additional evidence of the interplay of genetic, molecular, and cellular correlates governed by the Mx1 protein that critically determine disease outcome during lethal AI virus infection.

Cross-protective efficacies of highly-pathogenic avian influenza H5N1 vaccines against a recent H5N8 virus.

To investigate cross-protective vaccine efficacy of highly-pathogenic avian influenza H5N1 viruses against a recent HPAI H5N8 virus, we immunized C57BL/6 mice and ferrets with three alum-adjuvanted inactivated whole H5N1 vaccines developed through reverse-genetics (Rg): [Vietnam/1194/04xPR8 (clade 1), Korea/W149/06xPR8 (clade 2.2), and Korea/ES223N/03xPR8 (clade 2.5)]. Although relatively low cross-reactivities (10-40 HI titer) were observed against heterologous H5N8 virus, immunized animals were 100% protected from challenge with the 20 mLD50 of H5N8 virus, with the exception of mice vaccinated with 3.5μg of Rg Vietnam/1194/04xPR8. Of note, the Rg Korea/ES223N/03xPR8 vaccine provided not only effective protection, but also markedly inhibited viral replication in the lungs and nasal swabs of vaccine recipients within five days of HPAI H5N8 virus challenge. Further, we demonstrated that antibody-dependent cell-mediated cytotoxicity (ADCC) of an antibody-coated target cell by cytotoxic effector cells also plays a role in the heterologous protection of H5N1 vaccines against H5N8 challenge.

Poorly Differentiated Neuroendocrine Carcinoma in a Perigastric Lymph Node from an Unknown Primary Site

Neuroendocrine carcinomas from an unknown primary site are uncommon. The authors report on a case of neuroendocrine carcinoma in a perigastric lymph node (LN) with no primary site. A 52-year-old male patient with early gastric adenocarcinoma underwent treatment by endoscopic submucosal dissection, and, six months later, findings on a computed tomographic scan of the abdomen revealed a LN enlargement measuring 2.0 cm in the perigastric region. The patient underwent subtotal gastrectomy and regional LN dissection under a suggestive preoperative diagnosis of gastric adenocarcinoma with LN metastasis. However, microscopically, no residual tumor was found in the stomach, and the perigastric LN showed poorly differentiated neuroendocrine carcinoma (PDNEC). After an extensive workup, no primary site was identified. The patient also received four cycles of etoposide and cisplatin. Despite its extremely rare incidence, this case suggests that PDNEC of an unknown primary site is limited to a single site, and that resection should be considered in combination with chemotherapy.