RESUMO
AIM: To report positive predictive values (PPVs) of mammographic findings (MFs) of a screening cohort in Taiwan with a view to providing radiologists around the world with adequate information for assessing MFs before recommending biopsy for Asian women. MATERIALS AND METHODS: Between January 2014 and June 2017, 18,449 women received screening mammography at Tri-Service General Hospital (TSGH). Of these women, 1,622 exhibited specific MFs, namely mass (n=518), microcalcification (n=668), focal asymmetry (FA; n=462), and architectural distortion (AD; n=117). The distribution and PPVs of each MF were calculated after stratification based on cancer type, age, and breast density. RESULTS: The age group with the highest proportion of women was 50-59 years (48.1%), and most women presented with dense breasts (68.6%). The most common MF in the recalled women was microcalcification (41.2%) and the least common was AD (7.2%). AD was the most predictive MF for overall breast cancers, invasive carcinomas, and carcinomas in situ. Microcalcification was the second most predictive MF among recalled women for predicting overall breast cancers; however, it was less predictive than mass and FA in women who received a biopsy recommendation or underwent biopsy. CONCLUSION: AD can indicate the likelihood of breast cancer development in Asian women with abnormal screening results. Benign breast diseases are more likely to occur in women recommended for or receiving breast biopsy owing to microcalcification than to mass or FA.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Carcinoma in Situ/diagnóstico por imagem , Mamografia/métodos , Adulto , Fatores Etários , Idoso , Ásia , Mama/diagnóstico por imagem , Doenças Mamárias/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , TaiwanRESUMO
In animals, differentiation of germline from soma usually takes place during embryogenesis. Genes and their products that are preferentially expressed in the embryonic germ cells are regarded as candidates for maintaining germline fate or promoting germline identity. In Drosophila, for example, the protein encoded by the germline gene vasa is specifically restricted to the germ cells, while products of the gap gene hunchback (hb), a somatic gene, are preferentially expressed in the neuroblasts. In this study, we report the expression of both messenger RNA and protein encoded by Aphb, an hb orthologue in the asexual viviparous pea aphid Acyrthosiphon pisum, in germ cells as well as in neuroblasts. We infer that expression of Aphb messenger RNA in the germ cells during the formation of germaria is required for the anterior localization of Aphb in the protruding oocytes. Germarial expression and anterior localization of ApKrüppel was also identified but, unlike Aphb, its expression was not detected in the migrating germ cells. Very similar patterns of hb expression were also identified in the green peach aphid Myzus persicae, suggesting that germline expression of hb is conserved within the Aphididae. To date, this pattern of hb germline expression has not been reported in other insects.
Assuntos
Afídeos/metabolismo , Células Germinativas/metabolismo , Proteínas de Insetos/metabolismo , Animais , Afídeos/embriologia , Sequência de Bases , Proteínas de Ligação a DNA , Proteínas de Drosophila , Desenvolvimento Embrionário , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de TranscriçãoRESUMO
Previously we identified anterior localization of hunchback (Aphb) mRNA in oocytes and early embryos of the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum, suggesting that the breaking of anterior asymmetry in the oocytes leads to the formation of the anterior axis in embryos. In order to study posterior development in the asexual pea aphid, we cloned and analysed the developmental expression of caudal (Apcad), a posterior gene highly conserved in many animal phyla. We found that transcripts of Apcad were not detected in germaria, oocytes and embryos prior to the formation of the blastoderm in the asexual (viviparous) pea aphid. This unusual expression pattern differs from that of the existing insect models, including long- and short-germ insects, where maternal cad mRNA is passed to the early embryos and forms a posterior-anterior gradient. The first detectable Apcad expression occurred in the newly formed primordial germ cells and their adjacent blastodermal cells during late blastulation. From gastrulation onward, and as in other insects, Apcad mRNA is restricted to the posteriormost region of the germ band. Similarly, in the sexual (oviparous) oocytes we were able to identify anterior localization of Aphb mRNA but posterior localization of Apcad was not detected. This suggests that cad-driven posterior development is not conserved during early embryogenesis in asexual and sexual pea aphids.
Assuntos
Afídeos/embriologia , Desenvolvimento Embrionário , Proteínas de Homeodomínio/metabolismo , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Animais , Afídeos/genética , Afídeos/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Dados de Sequência Molecular , Ovário/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
To determine if double-stranded RNA (dsRNA) viruses exist and are potential virulence factors in Trichomonas gallinae, virus purification via ultracentrifugation was attempted for 12 T. gallinae isolates recovered from wild birds. Following purification, virus-like particles were not observed by transmission electron microscopy, nor were dsRNA segments visualized in agarose gels after electrophoresis of extracted RNA from any of the 12 T. gallinae isolates. However, virus particles and dsRNA segments were detected from a previously determined virus-infected T. vaginalis isolate as a control using identical purification procedures. Subsequent reverse transcription-polymerase chain reaction analysis of the dsRNA of the virus in this isolate revealed a novel sequence of the RNA-dependent RNA polymerase gene of T. vaginalis viruses.
Assuntos
Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/genética , Trichomonas/virologia , Animais , Aves/parasitologia , Análise por Conglomerados , Eletroforese em Gel de Ágar/métodos , Microscopia Eletrônica de Transmissão/métodos , Dados de Sequência Molecular , Filogenia , Vírus de RNA/classificação , RNA de Cadeia Dupla/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Trichomonas/isolamento & purificação , Ultracentrifugação/métodos , Vírion/ultraestruturaRESUMO
The purpose of this study is to investigate the clinical and histological features that may affect the survival of the patients and to evaluate the impact of post-operative adjuvant therapy on the outcomes of patients with stage IB and IIA carcinoma of the cervix. From August 1998 to January 2005, 140 patients with International Federation of Gynecology and Obstetrics stage IB and IIA cervical cancer were treated with radical hysterectomy and post-operative pelvic radiation therapy with or without chemotherapy. The median age was 55 years (range, 29-86 years). Seventy-six patients had stage IB and 64 patients had stage IIA disease. Tumour size was <4 cm in 96 patients and > or = 4 cm in 44 patients. One hundred and eleven patients had histology of squamous cell carcinoma, 12 patients has adenocarcinoma and 17 patients had other histologic types. Depth of stromal invasion was <2/3 in 20 patients and > or = 2/3 in 120 patients. Twenty-three patients had parametrial invasion and 117 patients had no parametrial invasion. Thirteen patients had lymphovascular space invasion and 127 had no lymphovascular space invasion. Nine patients had positive surgical margin and 131 patients had negative margin. Twenty-seven patients had pelvic lymph node metastasis and 113 patients had no pelvic lymph node metastasis. Seventy-five patients received concurrent chemoradiotherapy and 65 patients received radiotherapy alone. The 5-year overall survival (OAS) and disease-free survival were 83% and 72% respectively. In the log rank test, tumour size (P = 0.0235), pararmetrial invasion (P = 0.0121), pelvic lymph node metastasis (P < 0.0001) and adjuvant chemotherapy + radiotherapy (P = 0.0119) were significant prognostic factors for OAS, favouring tumour size <4 cm, absence of parametrial invasion and pelvic lymph node metastasis, and those who received adjuvant chemoradiotherapy. The patients who received radiation with concomitant chemotherapy had a 5-year OAS rate of 90% versus those who received radiotherapy alone, with a rate of 76%. For patients with high-risk early stage cervical cancer who underwent a radical hysterectomy and pelvic lymphadenectomy, adjuvant chemoradiotherapy resulted in better survival than radiotherapy alone. The addition of weekly cisplatin to radiotherapy is recommended. The treatment-related morbidity is tolerable.
Assuntos
Histerectomia , Neoplasias do Colo do Útero/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimioterapia Adjuvante/métodos , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Cuidados Pós-Operatórios , Prognóstico , Radioterapia Adjuvante , Fatores de Risco , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaRESUMO
Aberrant protein glycosylation could be a distinct surface-marker of cancer cells that influences cancer progression and metastasis because glycosylation can regulate membrane protein folding which alters receptor activation and changes epitope exposure for antibody (Ab) recognition. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a glycophosphoinositol-anchored protein, is a heavily glycosylated tumor antigen. However, the clinical significance and biological effect of CEACAM6 glycosylation has not been addressed in cancers. We recently developed an anti-CEACAM6 Ab (TMU) from an immune llama library which can be engineered to a single-domain (sd)Ab or a heavy-chain (HC)Ab. The TMU HCAb specifically recognized glycosylated CEACAM6 compared to the conventional antibodies. Using the TMU HCAb, we found that glycosylated CEACAM6 was a tumor marker associated with recurrence in early-stage OSCC (oral squamous cell carcinoma) patients. CEACAM6 promoted OSCC cell invasion, migration, cytoskeletal rearrangement, and metastasis via interaction with epidermal growth factor (EGF) receptor (EGFR) and enhancing EGFR activation, clustering and intracellular signaling cascades. These functions were modulated by N-acetylglucosaminyltransferase 5 (MGAT5) which mediated N-glycosylation at Asn256 (N256) of CEACAM6. Finally, the TMU sdAb and HCAb treatment inhibited the migration, invasion and EGF-induced signaling in CEACAM6-overexpressing cells. In conclusion, the complex N-glycosylation of CEACAM6 is critical for EGFR signaling of OSCC invasion and metastasis. Targeting glycosylated CEACAM6 with the TMU sdAb or TMU HCAb could be a feasible therapy for OSCC.
Assuntos
Antígenos CD/metabolismo , Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Bucais/patologia , Recidiva Local de Neoplasia/patologia , Adulto , Animais , Antígenos CD/genética , Asparagina/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/secundário , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Receptores ErbB/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Glicosilação , Humanos , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos SCID , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The dental follicle (DF), most often associated with unerupted teeth, is a condensation of ectomesenchymal cells that surrounds the tooth germ in early stages of tooth development. In the present study, we aim to isolate epithelial stem-like cells from the human DF and explore their potential differentiation into salivary gland (SG) cells. We demonstrated the expression of stem cell-related genes in the epithelial components of human DF tissues, and these epithelial progenitor cells could be isolated and ex vivo expanded in a reproducible manner. The human DF-derived epithelial cells possessed clonogenic and sphere-forming capabilities, as well as expressed a panel of epithelial stem cell-related genes, thus conferring stem cell properties (hDF-EpiSCs). When cultured under in vitro 3-dimensional induction conditions, hDF-EpiSCs were capable to differentiate into SG acinar and duct cells. Furthermore, transplantation of hDF-EpiSC-loaded native de-cellularized rat parotid gland scaffolds into the renal capsule of nude mice led to the differentiation of transplanted hDF-EpiSCs into salivary gland-like cells. These findings suggest that hDF-EpiSCs might be a promising source of epithelial stem cells for the development of stem cell-based therapy or bioengineering SG tissues to repair/regenerate SG dysfunction.
Assuntos
Saco Dentário/citologia , Células Epiteliais/citologia , Glândulas Salivares/citologia , Engenharia Tecidual/métodos , Animais , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Microscopia Confocal , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis. RESULTS: An IgA mAb called 6B8 was isolated from a library of mAbs reactive to surface proteins of T. vaginalis. The 6B8 mAb recognized a 44-kDa protein (TV44) by immunoblot analysis, and a full-length cDNA clone encoded a protein of 438 amino acids. Southern analysis revealed the gene (tv44) of T. vaginalis to be single copy. The tv44 gene was down-regulated at both the transcriptional and translational levels in iron-depleted trichomonads as well as in parasites after contact with immortalized MS-74 vaginal epithelial cells (VECs). Immunofluorescence on non-permeabilized organisms confirmed surface localization of TV44, and the intensity of fluorescence was reduced after parasite adherence to VECs. Lastly, an identical protein and gene were present in Tritrichomonas foetus and Trichomonas tenax. CONCLUSION: This is the first report of a T. vaginalis gene (tv44) encoding a surface protein (TV44) reactive with an IgA mAb, and both gene and protein were conserved in human and bovine trichomonads. Further, TV44 is independently down-regulated in expression and surface placement by iron and contact with VECs. TV44 is another member of T. vaginalis genes that are regulated by at least two independent signaling mechanisms involving iron and contact with VECs.
Assuntos
Ferro/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/metabolismo , Vagina/parasitologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Adesão Celular , Células Cultivadas , Regulação para Baixo , Células Epiteliais/parasitologia , Feminino , Regulação da Expressão Gênica , Genes de Protozoários , Humanos , Imunoglobulina A/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Alinhamento de Sequência , Transdução de Sinais , Trichomonas vaginalis/genética , Vagina/citologiaRESUMO
Physical processes in the gyrotron backward-wave oscillator (gyro-BWO) are investigated theoretically. Results indicate highly current-sensitive field profiles and hence sharply contrasting linear and saturated behaviors. The linear field extends over the entire structure length, whereas the saturated profile depends strongly on the energetics in the internal feedback loop. It is shown that this distinctive feature substantially influences the basic properties of the gyro-BWO including the start-oscillation current, efficiency, power scaling, and stability of tuning.
RESUMO
Measurements of the suppression of the yield per nucleon of J/psi and psi(') production for 800 GeV/ c protons incident on heavy nuclear targets, relative to light nuclear targets, have been made with very broad coverage in x(F) and p(T). The observed suppression is smallest at x(F) values of 0.25 and below, and increases at larger values of x(F). It is also strongest at small p(T). Substantial differences between psi(') and J/psi production are observed for the first time in p-A collisions. The suppression for psi(') production is stronger than that for J/psi for x(F) near zero, but becomes comparable to that for J/psi for x(F)>0.6.
RESUMO
T cell clones specific for hepatitis B core (HBcAg) and e (HBeAg) antigens of hepatitis B virus (HBV) were generated from liver infiltrates of HBeAg-positive patients. Analyzed with a panel of overlapping synthetic peptides spanning the complete sequences of HBcAg and HBeAg, eight clones responded specifically to the e2 peptide (PAYRPPNAPIL; amino acid residues 130-140 of HBcAg and HBeAg), which was doubly restricted by class I and II molecules. A preferential usage of the T cell receptor (TCR) alpha chain variable (V(alpha)) gene was found: V(alpha)12.1 for five HLA-Cw9(3)-restricted cytotoxic T lymphocyte (CTL) clones, and V(alpha)7.1 for three other HLA-DRw52-restricted type 1 helper T cell (Th1) clones. Although heterogeneous in the usage of TCR alpha chain joining region (J(alpha)) segments, their junctional-region sequences revealed conserved hydrophilic serine residues in seven of the eight e2-specific T cell clones. Single alanine substitution of the centrally located and the only hydrophilic asparagine residue of e2 peptide abrogated T cell responsiveness. The nonstimulatory e2 analogue could competitively inhibit e2-specific responses. These results demonstrate that both CTL and Th1 clones recognizing a determinant of HBcAg and HBeAg are present in the liver of chronic hepatitis B patients. The preferential V(alpha) gene usage and the expression of conserved residues in junctional-region sequences of TCRalpha chains by viral-peptide-specific, intrahepatic T cells may provide a T cell mechanism of HBV immunopathogenesis. Copyright 1994 S. Karger AG, Basel
RESUMO
Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.
Assuntos
Coloração Negativa/métodos , Vírus de RNA/ultraestrutura , Trichomonas vaginalis/virologia , Animais , Centrifugação com Gradiente de Concentração/métodos , Microscopia Eletrônica , RNA de Cadeia Dupla/ultraestrutura , Trichomonas vaginalis/crescimento & desenvolvimento , Vírion/ultraestruturaRESUMO
Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Approximately one-half of isolates of T. vaginalis are infected with a double-stranded (ds) RNA virus, which was described in the literature as a homogeneous population of icosahedral virus with isometric symmetry and 33 nm in diameter. The present study describes the heterogeneous virus population found in T. vaginalis isolate 347. This population comprises different virus sizes (33-200 nm) and shape (filamentous, cylindrical, and spherical particles). These observations were made in CsCl-purified virus fractions as well as the thin sections of parasites. Some viruses were only observed after slight changes in the technique where the sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The VLPs were found in the cytoplasm closely associated with the Golgi complex, with some VLPs budding from the Golgi, and other VLPs were detected adjacent to the plasma membrane. Unidentified cytoplasmic inclusions were observed in the region close to the VLPs and Golgi. These results indicate that T. vaginalis organisms may be infected with different dsRNA viruses simultaneously and suggest that T. vaginalis may be a reservoir for several viruses. We also showed some steps in the route of T. vaginalis virus and some aspects of the cytopathology of this infection. Purified VLPs were transfected to virus-free T. vaginalis isolates. Our results demonstrate that TVV attach and penetrate into trichomonads through endocytic coated pits and are maintained within vacuoles during batch culture for several daily passages. Immediately after virus transfection, many cells were lysed, whereas some intact reminiscent cells were recruited forming large clusters. Virus particles were found outside the cells, and in coated pits, within vacuoles in the cytoplasm, and infrequently within the nucleus. The Golgi complex showed changes in its electron density and in the cisternae structure. In lysed cells, virus particles were clearly seen over the residual membranes.
Assuntos
RNA de Cadeia Dupla/ultraestrutura , Trichomonas vaginalis/ultraestrutura , Trichomonas vaginalis/virologia , Vírion/ultraestrutura , Animais , Humanos , Microscopia EletrônicaRESUMO
Biomarkers that predict disease progression might assist the development of better therapeutic strategies for aggressive cancers, such as ovarian cancer. Here, we investigated the role of collagen type XI alpha 1 (COL11A1) in cell invasiveness and tumor formation and the prognostic impact of COL11A1 expression in ovarian cancer. Microarray analysis suggested that COL11A1 is a disease progression-associated gene that is linked to ovarian cancer recurrence and poor survival. Small interference RNA-mediated specific reduction in COL11A1 protein levels suppressed the invasive ability and oncogenic potential of ovarian cancer cells and decreased tumor formation and lung colonization in mouse xenografts. A combination of experimental approaches, including real-time RT-PCR, casein zymography and chromatin immunoprecipitation (ChIP) assays, showed that COL11A1 knockdown attenuated MMP3 expression and suppressed binding of Ets-1 to its putative MMP3 promoter-binding site, suggesting that the Ets-1-MMP3 axis is upregulated by COL11A1. Transforming growth factor (TGF)-beta (TGF-ß1) treatment triggers the activation of smad2 signaling cascades, leading to activation of COL11A1 and MMP3. Pharmacological inhibition of MMP3 abrogated the TGF-ß1-triggered, COL11A1-dependent cell invasiveness. Furthermore, the NF-YA-binding site on the COL11A1 promoter was identified as the major determinant of TGF-ß1-dependent COL11A1 activation. Analysis of 88 ovarian cancer patients indicated that high COL11A1 mRNA levels are associated with advanced disease stage. The 5-year recurrence-free and overall survival rates were significantly lower (P=0.006 and P=0.018, respectively) among patients with high expression levels of tissue COL11A1 mRNA compared with those with low expression. We conclude that COL11A1 may promote tumor aggressiveness via the TGF-ß1-MMP3 axis and that COL11A1 expression can predict clinical outcome in ovarian cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Colágeno Tipo XI/genética , Metaloproteinase 3 da Matriz/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Metaloproteinase 3 da Matriz/biossíntese , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/genética , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Proteína Proto-Oncogênica c-ets-1/genética , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Transdução de Sinais/genética , Proteína Smad2/biossíntese , Proteína Smad2/metabolismo , Taxa de Sobrevida , Fator de Crescimento Transformador beta1/farmacologia , Transplante Heterólogo , Resultado do TratamentoRESUMO
Cross-section values for Compton scattering on the proton were measured at 25 kinematic settings over the range s=5-11 and -t=2-7 GeV2 with a statistical accuracy of a few percent. The scaling power for the s dependence of the cross section at fixed center-of-mass angle was found to be 8.0+/-0.2, strongly inconsistent with the prediction of perturbative QCD. The observed cross-section values are in fair agreement with the calculations using the handbag mechanism, in which the external photons couple to a single quark.
RESUMO
Host parasitism by Trichomonas vaginalis is complex and in part mediated by adherence to vaginal epithelial cells (VECs). Four trichomonad surface proteins bind VECs as adhesins, and AP65 is a major adhesin with sequence identity to an enzyme of the hydrogenosome organelle that is involved in energy generation. In order to perform genetic analysis and assess the role of AP65 in T. vaginalis adherence, we silenced expression of ap65 using antisense RNA. The gene for ap65 was inserted into the vector pBS-neo in sense and antisense orientations to generate plasmids pBS-neoS (S) and pBS-neoAS (AS), respectively. Trichomonads were then transfected with S and AS plasmids for selection of stable transfectants using Geneticin, and the presence of plasmid in transfectants was confirmed by polymerase chain reaction of the neo gene. Reverse transcription polymerase chain reaction and Northern blot analysis showed decreased amounts of ap65 transcript in AS transfected parasites. Growth kinetics of the antisense-transfected and wild type organisms were similar, suggesting that silencing AP65 did not affect overall energy generation for growth. Immunoblot analysis using monoclonal antibody (mAb) to AP65 of AS transfectants showed decreased amounts of AP65 when compared to wild type or S transfectants. Not unexpectedly, this corresponded to decreased amounts of AP65 bound to VECs in a functional ligand assay. Reduction in parasite surface expression of AP65 was related to lower levels of adherence to VECs by AS-transfectants compared to control organisms. Antisense silencing of ap65 was not alleviated by growth of trichomonads in high iron, which up-regulates transcription of ap65. Our work reaffirms the role for AP65 as an adhesin, and in addition, we demonstrate antisense RNA gene silencing in T. vaginalis to study the contribution of specific genes in pathogenesis.
Assuntos
Moléculas de Adesão Celular/genética , Adesão Celular , Células Epiteliais/parasitologia , Regulação da Expressão Gênica , Inativação Gênica , Proteínas de Protozoários/genética , Trichomonas vaginalis/patogenicidade , Vagina/parasitologia , Animais , Linhagem Celular , Feminino , Humanos , Dados de Sequência Molecular , RNA Antissenso/genética , Trichomonas vaginalis/genética , Trichomonas vaginalis/crescimento & desenvolvimento , Trichomonas vaginalis/fisiologia , Vagina/citologiaRESUMO
Trichomonas vaginalis (TV) can be infected with double-stranded RNA (dsRNA) viruses that may have important implications for trichomonal virulence and disease pathogenesis. A cross-sectional study was conducted in a sexually transmitted diseases clinic to determine the prevalence and clinical significance of dsRNA viral infection of TV infecting men and women. Overall, dsRNA virus was present in 21 (75%) of 28 TV isolates (95% confidence interval [CI], 55%-89%). dsRNA viral infection of TV was not associated with the presence of discharge, dysuria, genital pruritus, or genital irritation or odor. However, patients with virus-positive isolates were significantly older than patients with virus-negative isolates (median age, 38 vs. 23 years; P=.003), and virus-positive isolates were more prevalent among women (19 [86%] of 22 isolates; 95% CI, 65%-97%) than among men (2 [33%] of 6 isolates; P=.02). The age and sex specificity of virus-positive isolates may aid in understanding the differences in chronicity and clinical presentation of TV in men and women.