In Vitro Anti-Cytomegalovirus Activity of Kampo (Japanese Herbal) Medicine

We examined the effect of three types of Kampo medicines on human cytomegalovirus (CMV) replication in the human embryonic fibroblast cell line, MRC-5. Treatment of cells with at least 0.01 μg/ml of Kampo medicines inhibited the cytopathic effects of CMV-infected MRC-5 cells. Moreover, Kampo medicine decreased the replication of CMV without affecting the inhibition of host cells, with a concomitant decrease in CMV DNA levels. However, Kampo medicine demonstrated no virocidal effect on cell-free CMV. Furthermore, western blotting analysis demonstrated that the Kampo medicine decreased the amount of 65 kDa late antigen expression in the infected cells. These results suggest that Kampo medicine may be sufficient to inhibit viral DNA replication and late protein synthesis, resulting in anti-CMV effects. Therefore, these three Kampo medicines have the potential of being a source of new powerful anti-CMV compounds

Human papillomavirus-16 presence and physical status in lung carcinomas from Asia

<p>Abstract</p> <p>Background</p> <p>Although human papillomavirus (HPV) genome has been detected in lung cancer, its prevalence is highly variable around the world. Higher frequencies have been reported in far-east Asian countries, when compared with European countries. The present study analysed the HPV-16 presence in 60 lung carcinomas from the Asian countries China, Pakistan and Papua New Guinea.</p> <p>Results</p> <p>HPV-16 was present in 8/59 (13%) samples. According to histological type, HPV-16 was detected in 8/18 (44%) squamous cell carcinomas (SQCs), which were mainly from Pakistan; 0/38 (0%) adenocarcinomas (ACs), which were mainly from China; and in 0/4 (0%) small cell carcinomas (SCLCs). The observed histological difference was statistically significant (p < 0.001). HPV-16 viral load was also determined using real-time polymerase chain reaction (qRT-PCR); it ranged between 411 to 2345 copies/100 ng of genomic DNA. HPV-16 genome was found integrated into the host genome in every HPV-16 positive carcinoma.</p> <p>Conclusion</p> <p>These results support the notion that HPV-16 infection is highly associated with SQCs in Pakistan. Our results show a frequent HPV-16 integration in SQCs, although the low viral load casts doubt respect a direct etiological role of HPV in lung carcinomas from Asia. Additional HPV-16 characterization is necessary to establish a direct or indirect etiological role of HPV in this malignancy.</p

Functional Interaction between Human Papillomavirus Type 16 E6 and E7 Oncoproteins and Cigarette Smoke Components in Lung Epithelial Cells

The smoking habit is the most important, but not a sufficient cause for lung cancer development. Several studies have reported the human papillomavirus type 16 (HPV16) presence and E6 and E7 transcripts expression in lung carcinoma cases from different geographical regions. The possible interaction between HPV infection and smoke carcinogens, however, remains unclear. In this study we address a potential cooperation between tobacco smoke and HPV16 E6 and E7 oncoproteins for alterations in proliferative and tumorigenic properties of lung epithelial cells. A549 (alveolar, tumoral) and BEAS-2B (bronchial, non-tumoral) cell lines were stably transfected with recombinant pLXSN vectors expressing HPV16 E6 and E7 oncoproteins and exposed to cigarette smoke condensate (CSC) at different concentrations. HPV16 E6 and E7 expression was associated with loss of p53 stability, telomerase (hTERT) and p16INK4A overexpression in BEAS-2B cells as demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). In A549 cells we observed downregulation of p53 but not a significant increase of hTERT transcripts. In addition, the HPV16 E6/E7 transfected cell lines showed an increased proliferation rate and anchorage-independent growth in a HPV16 E6 and E7 expression-dependent manner. Moreover, both HPV16 E6/E7 and mock transfected cells showed an increased proliferation rate and anchorage-independent growth in the presence of 0.1 and 10 µg/mL CSC. However, this increase was significantly greater in HPV16 E6/E7 transfected cells (p<0.001). Data were confirmed by FCSE proliferation assay. The results obtained in this study are suggestive of a functional interaction between tobacco smoke and HPV16 E6/E7 oncoproteins for malignant transformation and tumorigenesis of lung epithelial cells. More studies are warranted in order to dissect the molecular mechanisms involved in this cooperation

Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas

This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV+ lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV+ NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL

In vitro anti-viral activity of aqueous extracts of Kenyan Carissa edulis Prunus africana and Melia azedarach against human cytomegalovirus.

The aqueous extracts of three medicinal plants, Carissa edulis (Forssk.) Vahl (Apocynaceae), Prunus africana (Hook.f.) Kalkm (Rosaceae) and Melia azedarach L. (Meliaceae) have shown significant reduction in the replication of human cytomegalovirus (HCMV) in human embryonic lung (HEL) fibroblasts cells in vitro. Using the plaque inhibition assay for the determination of anti-viral activity, the HEL fibroblast cells cultured in 24 well plates were infected with 1 x 102 PFU 91S HCMV and treated with various concentrations of the extracts. The plaques formed were counted after 7 days incubation at 370C in 5% CO2 and the percent plaques inhibited were calculated against infected untreated control. The effective concentrations inhibiting plaque formation by 50% (EC50) was found between 40 to 80 &#956;g/ml for all the extracts. The cell cytotoxic concentrations (CC50) for each of the three extracts, by the trypan blue exclusion test, gave a safe therapeutic index. These results have demonstrated the potential anti-viral activities of the extracts of the three medicinal plants at non-cytotoxic concentrations. African Journal of Health Sciences Vol. 14 (3-4) 2007: pp. 143-14

Development of Ultra-Super Sensitive Immunohistochemistry and Its Application to the Etiological Study of Adult T-Cell Leukemia/Lymphoma

Antigen retrieval (AR) and ultra-super sensitive immunohistochemistry (ultra-IHC) have been established for application to archival human pathology specimens. The original ultra-IHC was the ImmunoMax method or the catalyzed signal amplification system (ImmunoMax/CSA method), comprising the streptavidin-biotin complex (sABC) method and catalyzed reporter deposition (CARD) reaction with visualization of its deposition. By introducing procedures to diminish non-specific staining in the original ultra-IHC method, we developed the modified ImmunoMax/CSA method with AR heating sections in an AR solution (heating-AR). The heating-AR and modified ImmunoMax/CSA method visualized expression of the predominantly simple present form of HTLV-1 proviral DNA pX region p40Tax protein (Tax) in adult T-cell leukemia/lymphoma (ATLL) cells in archival pathology specimens in approximately 75% of cases. The simple present form of Tax detected exhibited a close relation with ATLL cell proliferation. We also established a new simplified CSA (nsCSA) system by replacing the sABC method with the secondary antibody- and horse radish peroxidase-labeled polymer reagent method, introducing the pretreatments blocking non-specific binding of secondary antibody reagent, and diminishing the diffusion of deposition in the CARD reaction. Combined with AR treating sections with proteinase K solution (enzymatic-AR), the nsCSA system visualized granular immunostaining of the complex present form of Tax in a small number of ATLL cells in most cases, presenting the possibility of etiological pathological diagnosis of ATLL and suggesting that the complex present form of Tax-positive ATLL cells were young cells derived from ATLL stem cells. The heating-AR and ultra-IHC detected physiological expression of the p53 protein and its probable phosphorylation by Tax in peripheral blood mononuclear cells of peripheral blood tissue specimens from HTLV-1 carriers, as well as physiological and pathological expression of the molecules involved with G1 phase progression and G1–S phase transition (E2F-1, E2F-4, DP-1, and cyclin E) in ATLL and peripheral T-cell lymphoma cells. The ultra-IHC with AR is useful for etiological pathological diagnosis of ATLL since HTLV-1 pathogenicity depends on that of Tax, and can be a useful tool for studies translating advanced molecular biology and pathology to human pathology

Reconstrucción de la evolución molecular de la infección actual por el virus linfotrópico humano tipo I en Colombia.

The human T-lymphotropic virus type I (HTLV-I) infection is a public health problem in many endemic areas of Colombia. The subtyping of HTLV-I was based on the analysis of restriction fragment length polymorphisms (RFLP) in 3'LTR proviral DNA. From 31 HTLV-I isolates collected throughout Colombia, a RFLP analysis in a 737 bp 3'LTR fragment was performed. Fifty-eight percent (18/31) were identified as the Cosmopolitan subtype a, 19.4% (6/31) in the West African subtype b, 12.9% (4/31) in the Cosmopolitan subtype b and 9.6% (3/31) in the West African subtype c. The phylogenetic analysis of 3'LTR nucleotide sequences indicated that all the isolates in the current study were in the subgroup B or Japanese, in contrast with the highly divergent isolates from native Amerindians grouped in subgroup a or Transcontinental. The supported hypothesis was that of a post-Columbus introduction of virus represented in the African-American communities of the Colombian South Pacific. Some viral isolates from Colombian native Amerindians exhibited a nucleotide variation compatible with a Paleolithic introduction of the virus. The genetic diversity of HTLV-I in Colombia is complex and probably represents several independent introductions of lymphotropic virus.La infección por el virus linfotrópico humano tipo I (HTLV-I) es un problema de salud pública en varias zonas endémicas de Colombia. La subtipificación del HTLV-I se basa en los análisis de polimorfismos en el tamaño de fragmentos de restricción (RFLP) de la región 3?LTR del ADN proviral. A partir de 31 aislamientos de HTLV-I recolectados en diferentes regiones del territorio nacional se realizó un análisis de RFLP en un fragmento de ADN de 737 pb de la región LTR. El 58,1% (18/31) se incluyó dentro del subtipo Cosmopolita a; el 19,4% (6/31) en el Africano b; el 12,9% (4/31) en el Cosmopolita b, y el 9,6% (3/31) en el Africano c. Con base en análisis filogenéticos de secuencias nucleotídicas del 3?LTR, se demostró que los aislamientos colombianos incluidos en este trabajo se ubicaron dentro del subgrupo B o japonés, lo cual muestra gran divergencia con aquellos aislamientos de indígenas colombianos previamente reportados que se incluyeron dentro del subgrupo A o transcontinental. Nuestros datos apoyan la hipótesis de una introducción poscolombina del HTLV-I a Colombia que estaría representada en las comunidades negras de la costa del Pacífico del sur de Colombia que tuvieron ancestros africanos. Algunos aislamientos virales de indígenas colombianos mostraron una variación nucleotídica compatible con una introducción paleolítica. En su conjunto, los resultados obtenidos permiten postular que la actual diversidad genética del HTLV-I en Colombia es compleja y es el resultado de varios eventos de introducción, temporalmente separados

Expansión clónica y caracterización genómica del proceso de integración del virus linfotrópico humano tipo I en la leucemia/linfoma de células T en adultos

Introduction. Although the integration of human T-cell lymphotropic virus type I into the T-cells is not a random process, the mechanistic details are not understood.Objectives. The characteristics of the flanking host chromatin were evaluated at the integration sites in adult T-cell leukaemia/lymphoma (ATLL) patients infected with the virus.Materials and methods. From seven leukemic Colombian patients positive for the human T-cell lymphotropic virus type I (HTLV-I), lymphocyte DNA samples were extracted and amplified by inverse polymerase chain reaction (IPCR). Clonal expansion and human genome nucleotide composition in an extension of 50 bp was determined. To establish the characteristics of the human genome flanking provirus, 61 IPCR sequences from Colombian and Japanese ATLL patients, were analyzed in silico to obtain insights about the genomic structure, functions and nature of associated chromatin.Results. The clonal expansion of cell clones was predominantly oligoclonal. From 61 IPCR sequences, 155 alignments with homology higher than 95% (e-value &lt;0.05) were screened. Seventy-five percent of those sequences corresponded to non coding elements that include repetitive and non-repetitive DNA. Fifty percent of the proviral integrations were associated with chromosomes of A and B groups. Viral DNA integration tended to favor exons of genes that replicated early, controlled the cell cycle, or were involved in signal transduction.Conclusions. The results indicated that HTLV-I integration was preferentially directed towards genomic environments with high C:G content, and toward genes that replicate early, regulate cell cycle or involved with signal transduction.Introducción. Aunque la integración del virus linfotrópico humano tipo I no es al azar, se desconocen muchos de los detalles de este proceso.Objetivo. Evaluar las características de la cromatina celular adyacente a secuencias provirales en pacientes con leucemia/linfoma de células T en adultos asociada al virus.Materiales y métodos. Se extrajo el ADN de biopsias de siete pacientes colombianos con leucemia/linfoma de células T en adultos y positivos para el virus linfotrópico humano tipo I. &Eacute;ste se amplificó mediante reacción inversa en cadena de la polimerasa, para determinar el grado de expansión clónica y su composición de nucleótidos. A partir de 61 secuencias de ADN humano adyacentes a provirus, provenientes de pacientes leucémicos colombianos y japoneses, se efectuó un análisis in silico para obtener datos sobre su integración, las características de la cromatina y sus funciones asociadas.Resultados. La expansión de clones celulares fue predominantemente oligoclónica. De las 61 secuencias de ADN adyacente a provirus, se seleccionaron 155 alineamientos que cumplieron con los criterios de inclusión (homologías&ge;95%, e-value&le;0,05). De éstos, 74,84% fueron secuencias no codificantes repetidas y no repetidas. El 45,95% de las integraciones provirales se localizó en los cromosomas de los grupos A y B. Se observaron tendencias de integración hacia exones de genes que se replican tempranamente, regulan el ciclo celular y participan en la transducción de señales.Conclusiones. Los resultados permiten postular que la integración del virus linfotrópico humano tipo I se dirigiría hacia un ambiente genómico caracterizado por elevado contenido de C:G, genes de replicación temprana que regularían el ciclo celular y la transducción de señales

In vitro anti-viral activity of aqueous extracts of Kenyan Carissa edulis , Prunus africana and Melia azedarach against human cytomegalovirus

The aqueous extracts of three medicinal plants, Carissa edulis (Forssk.) Vahl (Apocynaceae), Prunus africana (Hook.f.) Kalkm (Rosaceae) and Melia azedarach L. (Meliaceae) have shown significant reduction in the replication of human cytomegalovirus (HCMV) in human embryonic lung (HEL) fibroblasts cells in vitro. Using the plaque inhibition assay for the determination of anti-viral activity, the HEL fibroblast cells cultured in 24 well plates were infected with 1 x 102 PFU 91S HCMV and treated with various concentrations of the extracts. The plaques formed were counted after 7 days incubation at 37\ub0C in 5% CO2 and the percent plaques inhibited were calculated against infected untreated control. The effective concentrations inhibiting plaque formation by 50% (EC50) was found between 40 to 80 \u3bcg/ml for all the extracts. The cell cytotoxic concentrations (CC50) for each of the three extracts, by the trypan blue exclusion test, gave a safe therapeutic index. These results have demonstrated the potential anti-viral activities of the extracts of the three medicinal plants at non-cytotoxic concentrations

Functional Interaction of Nuclear Domain 10 and Its Components with Cytomegalovirus after Infections: Cross-Species Host Cells versus Native Cells

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection