E2/E6 ratio and L1 immunoreactivity as biomarkers to determine HPV16-positive high-grade squamous intraepithelial lesions (CIN2 and 3) and cervical squamous cell carcinoma / 부인종양

OBJECTIVE: Human papillomavirus (HPV) 16 is the most carcinogenic HPV genotype. We investigated if HPV16 L1 capsid protein and E2/E6 ratio, evaluated by cervical cytology, may be used as biomarkers of ≥cervical intraepithelial neoplasia (CIN) 2 lesions. METHODS: Cervical specimens were obtained from 226 patients with HPV16 single infection. Using cytology specimen, L1 capsid protein and E2/E6 ratio were detected and the results were compared with those of the conventional histologic analysis of cervical tissues (CIN1–3 and squamous cell carcinoma [SCC]) to evaluate the association. RESULTS: The L1 positivity of CIN2/3 was significantly lower than that of normal cervical tissue (p < 0.001) and SCC demonstrated significantly lower L1 positivity than CIN1 (p < 0.001). The mean E2/E6 ratios of specimens graded as SCC (0.356) and CIN2/3 (0.483) were significantly lower than those of specimens graded as CIN1 (0.786) and normal (0.793) (p < 0.05). We observed that area under the receiver operating characteristic curve (AUC) for E2/E6 ratio (0.844; 95% confidence interval [CI]=0.793–0.895) was higher than that for L1 immunochemistry (0.636; 95% CI=0.562–0.711). A combination of E2/E6 ratio and L1 immunocytochemistry analyses showed the highest AUC (0.871; 95% CI=0.826–0.917) for the prediction of ≥CIN2 lesions. CONCLUSION: To our knowledge, this is the first study to validate HPV L1 capsid protein expression and decreased HPV E2/E6 ratio as valuable predictive markers of ≥CIN2 cervical lesions. Cervical cytology may be analyzed longitudinally on an outpatient basis with noninvasive procedures as against invasive conventional histologic analysis.

Programmed death-1 pathway blockade produces a synergistic antitumor effect: combined application in ovarian cancer / 부인종양

Programmed death-1 (PD-1) and its ligand are part of the immune checkpoint pathway that down-regulates effector T cells in immune response, thereby causing immune suppression. The PD-1/programmed death-ligand 1 (PD-L1) pathway can be blocked by antibodies to reverse tumor-mediated immunosuppression. However, advanced cancers such as stage III–IV ovarian cancer (OC) and certain types such as ID8 OC (a clone of C57BL/6 mouse OC) may hijack the PD-1/PD-L1 pathway to escape immune attack. When combined with chemotherapy, radiotherapy, targeted therapy, immunotherapy, or other agents, these PD-1/PD-L1 pathway blockages can produce a synergistic antitumor response in OC. Combined immunotherapy significantly prolongs overall survival by changing the tumor microenvironment through processes such as increasing the number of CD4⁺ or CD8⁺ T cells or cytokines in mice with OC and decreasing the number of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). OC patients treated with combined immunotherapy received better prognoses than those treated with monotherapy. This review reflects the move toward novel therapy combinations for OC and discusses these promising immunotherapeutic approaches, which are more cost-effective and effective than other approaches.

AAV-Mediated Astrocyte-Specific Gene Expression under Human ALDH1L1 Promoter in Mouse Thalamus

Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter. To study the function of astrocytes in thalamus, which serves as a relay station, there is a great need for identifying an alternative astrocyte-specific promoter in thalamus. Recently, a new astrocyte-specific promoter of ALDH1L1 has been identified. However, it has not been examined in thalamus. Here we developed and characterized an AAV vector expressing Cre recombinase under the human ALDH1L1 promoter, AAV-hALDH1L1-Cre. To test the cell-type specific expression of AAV-hALDH1L1-Cre, AAV virus was injected into several brain regions of Ai14 (RCL-tdTomato) mouse, which reports Cre activity by tdTomato expression. In thalamus, we observed that tdTomato was found mostly in astrocytes (91.71%), with minimal occurrence in neurons (2.67%). In contrast, tdTomato signal was observed in both neurons and astrocytes of the amygdala (neuron: 68.13%, astrocyte: 28.35%) and hippocampus (neuron: 76.25%, astrocyte: 18.00%), which is consistent with the previous report showing neuronal gene expression under rat ALDH1L1 promoter. Unexpectedly, tdTomato was found mostly in neurons (91.98%) with minimal occurrence in astrocytes (6.66%) of the medial prefrontal cortex. In conclusion, hALDH1L1 promoter shows astrocyte-specificity in thalamus and may prove to be useful for targeting thalamic astrocytes in mouse.

Immunohistochemistry and Polymerase Chain Reaction for Detection Human Papilloma Virus in Warts: A Comparative Study

BACKGROUND: Immunohistochemistry and polymerase chain reaction (PCR) are the most widely used methods for the detection of viruses. PCR is known to be a more sensitive and specific method than the immunohistochemical method at this time, but PCR has the disadvantages of high cost and skilled work to use widely. With the progress of technology, the immunohistochemical methods used in these days has come to be highly sensitive and actively used in the diagnostic fields. OBJECTIVE: To evaluate and compare the usefulness of immunohistochemistry and PCR for detection human papilloma virus (HPV) in wart lesions. METHODS: Nine biopsy samples of verruca vulgaris and 10 of condyloma accuminatum were examined. Immunohistochemical staining using monoclonal antibody to HPV L1 capsid protein and PCR were done for the samples. DNA sequencing of the PCR products and HPV genotyping were also done. RESULTS: HPV detection rate was 78.9% (88.9% in verruca vulgaris, 70.0% in condyloma accuminatum) on immunohistochemistry and 100.0% for PCR. HPV-6 genotype showed a lower positivity rate on immunohistochemistry (50.0%) as compared to that of the other HPV genotypes. CONCLUSION: Immunohistochemistry for HPV L1 capsid protein showed comparable sensitivity for detection HPV. Considering the high cost and great effort needed for the PCR methods, we can use immunohistochemistry for HPV L1 capsid protein with the advantage of lower cost and simple methods for HPV detection.

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 % de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico. La coincidencia entre el método inmunoquímico y la RCP-TR fue de un 98,6 por ciento, la sensibilidad fue de un 98,57 por ciento y la especificidad de un 91,67 por ciento, con valores predictivos negativo y positivo por encima del 90 por ciento. Conclusiones: se demostró la validez del método inmunoquímico como prueba confirmatoria de la infección por PVH 16. Dicho método probó ser sensible, sencillo y no requiere de una compleja infraestructura para detectar PVH 16 en muestras cervicales. Además, esta técnica permite obtener información rápidamente y evita el uso de métodos invasivos(AU)

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific. The diagnostic agreement between the immunochemical method and the real-time polymerase chain reaction reached 98.6 percent, sensitivity was 98.57 percent and specificity was 91.67 %, with positive and negative predictive values above 90 percent. Conclusions: the validity of the immunochemical method as a confirmatory test for infection by HPV-16 has been demonstrated. The normalized immunochemical method proved to be a sensitive, simple, relatively fast method to detect HPV from clinical samples of cervical cells. Furthermore, this method provides information quickly, avoiding the use of invasive methods in patients(AU)

MAD2 Expression in Ovarian Carcinoma: Different Expression Patterns and Levels among Various Types of Ovarian Carcinoma and Its Prognostic Significance in High-Grade Serous Carcinoma

BACKGROUND: Mitotic arrest deficiency protein 2 (MAD2) is a key component of spindle assembly checkpoint function, which mediates cell apoptosis through microtubule kinetics. Aberrant expression of MAD2 is believed to be associated with the development of chromosome instability. MAD2 also has a signihicant role in cellular drug resistance to taxane chemotherapeutic agents. METHODS: Expression of MAD2 and p53 was investigated using immunohistochemistry in 85 cases of ovarian carcinomas. Clinicopathological data including progression-free survival were analyzed. RESULTS: A significant (p=.035) association was observed between the grade of serous carcinoma and the expression level of MAD2. While low-grade serous carcinoma showed a low-level expression of MAD2, high-grade serous carcinoma showed a high-level expression of MAD2. We also determined that low-level expression of MAD2 was associated with reduced progression-free survival (PFS) (p=.016) in high-grade serous carcinoma. CONCLUSIONS: MAD2 expression in ovarian carcinoma is related to the grade of serous carcinoma and PFS of high-grade serous carcinoma. Expression level of MAD2 detected by immunohistochemistry may serve as an indicator in predicting the response of microtubule-interfering chemotherapeutic agents.

Molecules and their functions in autophagy

Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.

Biological status and L1 protein expression of human papillomavirus 16 in patients with cervical lesions / 中华临床感染病杂志

Objective To evaluate the relationship of biological status of HPV 16 and expression of L1 protein with the degree of cervical tumorigenesis.Methods Sixty-one patients with cervical lessions were enrolled and divided into five groups according to pathology of cervical lesions,including chronic cervicitis(n = 27),cervical intraepithelial neoplasia(CIN) Ⅰ-Ⅱ(n = 10),carcinoma in situ(n = 8),early-stage(n =7)and mid/late-stage(n = 9)of cervical carcinoma.HPV type and its biological status were detected by PCR amplification,and L1 protein in HPV 16 positive tissues was detected by Western blot.Kruskal-Wallis was used to compare between the groups,and Pearson correlation analysis was used to evaluate the relationship of HPV biological status and L1 protain expression with cervical lesions.ResultsFifty-three out of 61 patients with cervical lesions were detected with HPV positive(86.9%).All 25 patients with chronic cervicitis were in HPV free mode; in CIN Ⅰ-Ⅱ group,6 patients were in free and 2 in integral mode; in carcinoma in situ group,1 patient in free,3 in mixed and 3 in integral mode; in the early-stage cervical cancer group,2 patients in mixed and 4 in integral mode; in mid/late-stage cancer group,1 patient in mixed and 6 in integral mode.A strong positive correlation was found between the HPV in integral mode and the severity of cervical lesions(r = 0.705,P ＜0.01).The expression of L1 protein was negatively correlated with the aggravation of cervical lesions in HPV 16-positive patients(r = -0.755,P ＜0.01).Conclusion The integral mode of HPV16 and low expression of L1 protein may have predictive value for the severity of cervical lesions.

The roles of YKL-40 in atherosclerosis / 国际脑血管病杂志

YKL-40 (human cartilage glycoprotein 39) is a newly discovered inflammatory cytokine, which belongs to the member of 18 glycosyl hydrolase of mammal family. Previous studies have indicated that YKL-40 is associated with the acute or chronic inflammatory diseases and tumors. Studies in recent years have suggested that YKL-40 may be involved in the occurrence and development of atherosclerotic plaques, and it is correlated with the plaque instability. The physiological function and the mechanisms of YKL-40 are not fully understood. It may have the roes of promoting vascular smooth muscle migration and proliferation, promoting cell adhesion and proliferation, as well as regulating extracellular matrix remodeling The detection of YKL-40 may have some significance in the aided diagnosis, predicting prognosis, prevention of cardiocerebrovascular diseases, and even the establishment of new therapeutic strategies.

Role of breast regression protein-39/YKL-40 in asthma and allergic responses

BRP-39 and its human homolog YKL-40 have been regarded as a prototype of chitinase-like proteins (CLP) in mammals. Exaggerated levels of YKL-40 protein and/or mRNA have been noted in a number of diseases characterized by inflammation, tissue remodeling, and aberrant cell growth. Asthma is an inflammatory disease characterized by airway hyperresponsiveness and airway remodeling. Recently, the novel regulatory role of BRP-39/YKL-40 in the pathogenesis of asthma has been demonstrated both in human studies and allergic animal models. The levels of YKL-40 are increased in the circulation and lungs from asthmatics where they correlate with disease severity, and CHI3L1 polymorphisms correlate with serum YKL-40 levels, asthma and abnormal lung function. Animal studies using BRP-39 null mutant mice demonstrated that BRP-39 was required for optimal allergen sensitization and Th2 inflammation. These studies suggest the potential use of BRP-39 as a biomarker as well as a therapeutic target for asthma and other allergic diseases. Here, we present an overview of chitin/chitinase biology and summarize recent findings on the role of BRP-39 in the pathogenesis of asthma and allergic responses.

Expression of HPV L1 protein and p16 in cervical lesions / 临床与实验病理学杂志

Purpose To study the expression of HPV L1 protein and p16 in various cervical lesions and to explore the value of HPV L1 protein and p16 immunostaining in predicting the progression from CIN1 to CIN3 and squamous cell carcinoma (SCC).Methods Expression of HPV L1 protein and p16 in 18 cases of CIN1, 9 cases of CIN2, 8 cases of CIN3 and 6 cases of SCC was detected by immunohistochemistry.Results The average positive rates of HPV L1 protein in cervical lesions were 26.8%, and HPV L1 protein was positive in 38.9% of CIN1 and 44.4% of CIN2, but in 0 of CIN3 and SCC. In contrast, the average positive rate of p16 protein in cervical lesions was 68.3%, p16 protein was positive in 38.9% of CIN1and 77.8% of CIN2, but in 100% of CIN3 and SCC. p16-/HPV L1+ and p16-/HPV L1- cases represented 61.1% of CIN1, but 0 of CIN3 and SCCs, whereas p16+/HPV L1- cases represented 100% of CIN3 and SCC.Conclusions Expression of HPV L1 protein decreases whereas p16 protein increases with lesion progression. p16+/HPV L1- cases have the potential for progression, whereas p16-/HPV L1+ and p16-/HPV L1- cases may not be progressive lesions or potentially in remission.

Mutation analysis of p31(comet) gene, a negative regulator of Mad2, in human hepatocellular carcinoma

Failure of mitotic checkpoint machinery leads to the chromosomal missegregation and nuclear endoreduplication, thereby driving the emergence of aneuploidy and tetraploidy population. Although abnormal nuclear ploidy and the resulting impairment of mitotic checkpoint function are typical physiological event leading to human hepatocellular carcinoma, any mutational change of mitotic checkpoint regulators has not yet been discovered. Therefore, we investigated the mutation of p31(comet), a recently identified mitotic checkpoint regulator, in human hepatocellular carcinoma. Of 51 human hepatocellular carcinoma tissue and 6 cell lines tested, five samples exhibited nucleotide sequence variations dispersed on four sites within the entire coding sequence. Among these sites with sequence substitutions, three were found to be missense mutation accompanied with amino acid change but one was a silent mutation. Of these sequence substitutions, two were present in both tumor and non-tumor liver tissues, suggesting the possibility of polymorphism. The present findings indicate that p31(comet) does not have an impact on the formation of aneuploidy and tetraploidy found in human hepatocellular carcinoma.