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2.
Virology ; 524: 106-113, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30170240

RESUMO

The major capsid protein of HPV, L1, assembles into pentamers that form a T = 7 icosahedral particle, but the location of the co-assembled minor capsid protein, L2, remains controversial. Several researchers have developed useful monoclonal antibodies targeting L2, but most react with linear epitopes toward the N-terminus. As a means to better define the virus capsid and better assess the localization and exposure of L2 epitopes in the context of assembled HPV, we have developed a panel of 30 monoclonal antibodies (mAbs) which target the N-terminus of L2 amino acids 11-200, previously defined as a broadly protective immunogen. Select mAbs were processed with enzymes and anti-L2 Fabs were generated. These new mAb/Fab probes will be beneficial in future studies to unravel the placement of L2 and to help better define the role of L2 in the HPV lifecycle and the nature of the broadly protective epitopes.


Assuntos
Alphapapillomavirus/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/virologia , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Epitopos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Vírion
3.
Cancer Res ; 48(4): 943-9, 1988 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-2962735

RESUMO

An antiproliferative suppressor lymphokine was produced from rat T-cells specifically in response to the poorly immunogenic syngeneic mammary adenocarcinoma 13762A. The tumor-induced suppressor lymphokine (TISL) was produced late in culture (peak production on Days 4 and 5) and showed strong but selective inhibitory activity on a variety of immune responses. The immune peritoneal exudate cell response to a highly immunogenic clone from the parental tumor (clone 18A) and the concanavalin A-stimulated response of nonimmune spleen cells were inhibited strongly by TISL. In contrast, the immune spleen cell response to 13762A and the lipopolysaccharide response of nonimmume spleen cells were unaffected. Preliminary molecular weight and physicochemical analysis of TISL indicated that the molecule was large (Mr greater than 350,000); partially sensitive to 75 degrees C treatment for 15 min and to pH 2.0 treatment; only partly degraded by the enzymes trypsin, chymotrypsin, and proteinase K; and completely destroyed by boiling. Although TISL was produced specifically in response to 13762A tumor, prior immunization in vivo was not necessary for the induction of the suppressor lymphokine. These results indicate that populations of rat lymphocytes contain naturally occurring TISL secreting cells, which can be activated specifically by tumor antigens such as those expressed by 13762A.


Assuntos
Adenocarcinoma/imunologia , Citotoxicidade Imunológica , Neoplasias Mamárias Experimentais/imunologia , Fatores Supressores Imunológicos/biossíntese , Linfócitos T Reguladores/imunologia , Animais , Anticorpos/imunologia , Proteínas do Sistema Complemento/imunologia , Feminino , Ativação Linfocitária , Ratos , Ratos Endogâmicos F344
4.
J Mol Biol ; 259(2): 249-63, 1996 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-8656427

RESUMO

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.


Assuntos
Proteínas do Capsídeo , Capsídeo/ultraestrutura , Papillomaviridae/ultraestrutura , Polyomavirus/ultraestrutura , Animais , Antígenos Virais/química , Papillomavirus Bovino 1/química , Papillomavirus Bovino 1/ultraestrutura , Capsídeo/química , Papillomavirus de Coelho Cottontail/química , Papillomavirus de Coelho Cottontail/ultraestrutura , Humanos , Papillomaviridae/química , Polyomavirus/química , Coelhos , Alinhamento de Sequência , Vírus 40 dos Símios/química , Vírus 40 dos Símios/ultraestrutura , Proteínas Estruturais Virais/química
5.
J Invest Dermatol ; 101(2): 227-31, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8393903

RESUMO

This study analyzed in vivo antiviral cellular immune reactions in the Shope rabbit papilloma-carcinoma model. Antigens studied in experimentally infected domestic rabbits were cottontail rabbit papillomavirus particles produced with the athymic (nu/nu) mouse xenograft system and bacterial fusion proteins containing the major or minor capsid protein. Recall reactions to antigens were tested by classic intracutaneous tests. Positive reactions had a biphasic course. Histopathology of skin test biopsy specimens showed infiltrating polymorphonuclear cells during the early stages. Later they were replaced by predominantly perivascular infiltrates composed of mononuclear cells. Time course of swelling and infiltrates resembled a delayed-type hypersensitivity reaction. Ten of 11 regressor rabbits (p = 0.00006) and 10 of 20 progressors (p = 0.009) had positive skin tests with intact and/or denaturated virus particles and individual capsid proteins also could elicit specific skin reactions. Skin reactivity to the cottontail rabbit papillomavirus particles was also greater (p = 0.042) in regressor rabbits (8 of 11) when compared to progressors (7 of 20). Recall reactions remained detectable at post-regression times, ranging from several months up to more than 2 years. We conclude that specific skin reactions against the cottontail rabbit papillomavirus in infected domestic rabbits exist, and are strongly positive to intact particles of this papillomavirus in animals (regressors) clinically free of disease.


Assuntos
Antígenos Virais/administração & dosagem , Papillomavirus de Coelho Cottontail/imunologia , Testes Cutâneos , Infecções Tumorais por Vírus/imunologia , Animais , Antígenos Virais/genética , Biópsia , Modelos Animais de Doenças , Imunidade , Proteínas/análise , Coelhos , Pele/patologia , Neoplasias Cutâneas/imunologia
6.
J Invest Dermatol ; 99(6): 813-8, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1469296

RESUMO

The objective of the present study was to assess the utility of Shope rabbit papillomas as an animal model system for studying topical podofilox treatment and to evaluate dose-response relations and influence of duration of papilloma growth prior to treatment. New Zealand White rabbits received inoculations of cottontail rabbit papillomavirus (CRPV) virions of two dilutions at four sites total on the dorsum. Two papillomas on the left side were treated with podofilox (Oclassen Pharmaceuticals, Inc., San Rafael, CA). The drug was given topically twice each day, 5 d per week, for 21 d. We evaluated the effects of drug dose and the duration of papilloma growth prior to treatment. Results indicated that treatment beginning on day 28 with both 0.5 and 2.5% (w/v) podofilox inhibited papilloma growth, but 5.0% was more effective. In a separate experiment, papillomas were treated at 7, 21, or 60 d after virus inoculation. At 7 d, the untreated lesions were latent (not visible). At 21 d after infection, they were about 2.5 mm in diameter. At 60 d, papillomas were about 25 mm. Treatment with 5.0% podofilox beginning on any of those days strongly inhibited papilloma growth. Neither Southern blots nor PCR detected CRPV DNA in cured sites of previous virus infection. Antibody production to CRPV virion was not affected by drug treatment. 5.0% podofilox irritated normal skin adjacent to papillomas as evidenced by inflammation, induration, and superficial erosion. However, healing was satisfactory and no scarring resulted. We concluded that the Shope papilloma was a good model system for studying podofilox treatment because the lesions responded to drug across a broad range of drug concentrations and papilloma sizes.


Assuntos
Papiloma/tratamento farmacológico , Podofilotoxina/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Administração Tópica , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Podofilotoxina/administração & dosagem , Coelhos , Fatores de Tempo
7.
J Invest Dermatol ; 105(3): 438-44, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7665926

RESUMO

Human papillomavirus type 11 (HPV-11), produced from the athymic mouse xenograft system, was shown to infect cultured neonatal human foreskin keratinocytes and the HaCaT keratinocyte cell line in vitro. Infection was documented by the appearance of HPV-11-specific spliced mRNA, detected by reverse transcriptase-polymerase chain reaction. Purified HPV-11 virions at concentrations of approximately 10(7) particles/ml could successfully evoke infection in this system. Infection was completely abrogated by preincubation of the HPV-11 inoculum with mouse anti-HPV-11 monoclonal antibodies, experimentally immunized animal sera, or sera of human patients with HPV infection. Concurrent detection of cellular mRNA for the beta-actin gene, also by reverse transcriptase-polymerase chain reaction, provided an internal control confirming RNA recovery and successful reverse transcriptase-polymerase chain reaction. Using this approach, it was possible to determine semiquantitative titers for test solutions of HPV-11-neutralizing antibodies. The in vitro system for HPV-11 infectivity and neutralization may be useful in the study of the immune response to HPV-11 infection or immunization in patients.


Assuntos
Anticorpos/imunologia , Testes de Neutralização , Papillomaviridae/imunologia , Papillomaviridae/fisiologia , Actinas/genética , Anticorpos Monoclonais , Células Cultivadas , Humanos , Papillomaviridae/genética , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Viral/análise , Transcrição Gênica
8.
Clin Exp Metastasis ; 18(8): 683-93, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11827072

RESUMO

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.


Assuntos
Neoplasias Pulmonares/prevenção & controle , Neoplasias Mamárias Experimentais/prevenção & controle , Proteínas de Neoplasias , Proteínas/fisiologia , Animais , Northern Blotting , Southern Blotting , Primers do DNA/química , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Fosforilação , RNA Mensageiro/metabolismo , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas/metabolismo
9.
J Immunol Methods ; 66(2): 357-68, 1984 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-6418824

RESUMO

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.


Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Técnica de Placa Hemolítica , Linfócitos T Citotóxicos/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Células Cultivadas , Fluoresceína-5-Isotiocianato , Fluoresceínas , Antígenos H-2/genética , Hibridomas/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Células-Tronco/imunologia , Tiocianatos
10.
Neuroscience ; 58(3): 605-25, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7513388

RESUMO

The anterograde and retrograde transport of wheat germ agglutinin-horseradish peroxidase was used to study the trigeminoperibrachial pathway in the muskrat after injections of tracer into either the medullary dorsal horn or the dorsolateral pons. After injections into the medullary dorsal horn, labeled fibers ascended into the ipsilateral dorsolateral pons via the spinal trigeminal tract, within the neuropil of the trigeminal sensory complex and within the reticular formation adjacent to the spinal trigeminal nucleus. At caudal levels of the ipsilateral peribrachial area, dense terminal-like label distributed in the Kölliker-Fuse nucleus continued into the lateral parabrachial nucleus. At intermediate levels ipsilaterally, the Kölliker-Fuse nucleus again was labeled densely, as were areas analogous to the external lateral and external medial subnuclei of the parabrachial nucleus in the rat. A thin band of label along the ventral spinocerebellar tract outlined an unlabeled area in the central portion of the lateral parabrachial nucleus. Rostrally near the pontomesencephalic junction, the area designated the superior lateral subnucleus in the hamster was labeled, while sparser label was present more dorsally. Contralateral to the injections, caudal and intermediate levels of the peribrachial area contained only scant reaction product. However, the rostral area of the superior lateral subnucleus was labeled densely via fibers ascending in the trigeminothalamic tract. Injections made just rostral to the obex and either centered in or including the dorsal or ventral paratrigeminal nuclei produced similar labeling at caudal and intermediate levels of the peribrachial area. An exception, however, was that the caudal medial parabrachial nucleus was also labeled after the dorsal paratrigeminal injection. Also, only scant label was found in the rostral third of the dorsolateral pons on either side after these injections. Both trigeminothalamic and trigeminolemniscal pathways were labeled contralaterally after these injections. These trigeminal projections to the dorsolateral pons were compared to the projections from the nucleus tractus solitarii and the ventrolateral medulla. Numerous trigeminal neurons were labeled retrogradely after injections of wheat germ agglutinin-horseradish peroxidase into the dorsolateral pons. In the medullary dorsal horn, they were found almost exclusively in laminae I and V. Labeled neurons in lamina I were especially prominent in rostral ventral levels of the medullary dorsal horn. Labeled cells in lamina I were continuous with others found in the displaced band of substantia gelatinosa at the interface of the subnucleus caudalis and subnucleus interpolaris, as well as with those found in the ventral and dorsal paratrigeminal nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Arvicolinae/fisiologia , Ponte/fisiologia , Gânglio Trigeminal/fisiologia , Animais , Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Histocitoquímica , Peroxidase do Rábano Silvestre , Bulbo/citologia , Bulbo/fisiologia , Fibras Nervosas/fisiologia , Vias Neurais/citologia , Vias Neurais/fisiologia , Ponte/citologia , Gânglio Trigeminal/citologia , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano Silvestre , Aglutininas do Germe de Trigo
11.
Virus Res ; 21(3): 169-79, 1991 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1722596

RESUMO

Monoclonal antibodies were generated against cottontail rabbit papillomavirus (CRPV) and tested for neutralization of CRPV-induced papillomas on domestic NZW rabbits. Intact CRPV was semi-purified on CsCl gradients and used to immunize BALB/c mice. Hybridomas were prepared from a fusion with lymph node cells, and supernatants from growing hybridomas were analyzed by enzyme-linked immunosorbent assay (ELISA) for reactivity to both intact and disrupted CRPV virion antigen. Supernatants from 22 cultures were initially selected that were responsive to CRPV. Ten were reactive to intact CRPV alone, 4 were reactive only to disrupted CRPV, and 8 were reactive to both intact and disrupted CRPV virion antigen. None of these supernatants contained antibodies which recognized epitopes on CRPV capsid proteins (L1 and L2) that were separated on Western blots. Five hybridomas which produced antibodies that bound to intact CRPV, and did not react to intact HPV-11 or BPV-1 were selected and tested for antibody-mediated neutralization of CRPV infectivity. All five monoclonal antibodies were neutralizing, and identified epitopes on intact CRPV virions which were non-linear and conformational in nature. The five neutralizing monoclonal antibodies appeared to recognize a similar epitope or epitope cluster on the intact CRPV virion as determined by competition ELISA.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Papillomaviridae/imunologia , Infecções Tumorais por Vírus/prevenção & controle , Animais , Anticorpos Monoclonais/biossíntese , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Cinética , Testes de Neutralização , Papillomaviridae/crescimento & desenvolvimento , Conformação Proteica , Coelhos , Análise de Regressão , Infecções Tumorais por Vírus/microbiologia
12.
Virus Res ; 28(2): 195-202, 1993 May.
Artigo em Inglês | MEDLINE | ID: mdl-7686316

RESUMO

Mouse monoclonal antibodies were generated against intact infectious BPV-1 virions by methods previously described (Christensen et al., 1990). ELISA was used to screen for reactivities to intact and/or disrupted BPV-1, CRPV and HPV-11 virions. Several hybridomas were initially selected that showed antibody reactivity by ELISA to both intact and disrupted BPV-1, to disrupted BPV-1 only, or to intact BPV-1 virions. One monoclonal antibody, designated B1.A1, which reacted only to intact BPV-1 was selected for virus neutralization analyses. ELISA demonstrated that this monoclonal antibody bound to intact BPV-1 virions, but not to intact CRPV, HPV-11 or to disrupted papillomavirus (PV) antigens. Strong neutralization of BPV-1-induced focus formation of mouse C127 cells by monoclonal B1.A1 was observed. The neutralization titer was equivalent to the neutralization titer obtained with a polyclonal rabbit anti-BPV-1 virion antisera, and directly correlated with antibody concentration as determined by ELISA. These results extend our previous analyses on the epitopes of infectious papillomaviruses as defined by monoclonal antibodies that identify neutralizing epitopes. The nature of these epitopes is such that maintenance of the quaternary structure of the infectious virions is necessary for preservation of the antigenicity of the neutralizing epitope.


Assuntos
Papillomavirus Bovino 1/imunologia , Epitopos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Camundongos , Testes de Neutralização
13.
Antiviral Res ; 48(2): 131-42, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11114415

RESUMO

A series of nucleoside analogues were tested for in vivo anti-papillomavirus activity using the cottontail rabbit papillomavirus (CRPV) domestic rabbit model. Compounds were delivered either topically, injected into growing papillomas, or delivered subcutaneously at a site remote from the papillomas. Compounds tested included cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (HPMPC); cyclic HPMPC (cHPMPC); cyclopentenylcytosine (CPE-C); lobucavir [1R(1alpha,2beta,3alpha)]-9-[2, 3-bis(hydroxymethyl)cyclobutyl]guanine; 9-((2-phosphonylmethoxy)propyl)adenine (PMPA); adefovir 9-((2-phosphonylmethoxy)ethyl)adenine(PMEA) and cyclopropyl 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (cyclopropylPMEDAP). Dose response curves and time-course treatments were included for most compounds tested. Strong anti-viral activity was detected using cidofovir and cHPMPC when delivered either topically or by the intralesional route. Complete cures were obtained using 1% (w/v) topical cidofovir at dosing schedules of twice daily for 8 weeks beginning at 4 weeks after CRPV infection, which represents a time when papillomas were clearly visible. Complete cures of large established papillomas were obtained by intralesional injection of 1% cidofovir three times per week for 8 weeks. Topical treatments with adefovir had strong anti-viral activity, cyclopropyl PMEDAP had moderate anti-viral activity, and CPE-C, PMPA and lobucavir showed no effects. These data indicate that certain nucleoside analogues have strong in vivo anti-papillomavirus activity and that the CRPV/rabbit model is a good model for assessing clinical responses of anti-viral treatments for patients with HPV disease.


Assuntos
Antivirais/uso terapêutico , Papillomavirus de Coelho Cottontail , Citosina/uso terapêutico , Organofosfonatos , Compostos Organofosforados/uso terapêutico , Papiloma/tratamento farmacológico , Infecções por Papillomavirus/tratamento farmacológico , Animais , Cidofovir , Papillomavirus de Coelho Cottontail/efeitos dos fármacos , Papillomavirus de Coelho Cottontail/patogenicidade , Citosina/análogos & derivados , Modelos Animais de Doenças , Humanos , Nucleosídeos/química , Nucleosídeos/uso terapêutico , Papiloma/virologia , Infecções por Papillomavirus/virologia , Coelhos , Resultado do Tratamento , Infecções Tumorais por Vírus/diagnóstico , Infecções Tumorais por Vírus/tratamento farmacológico
14.
J Virol Methods ; 39(1-2): 207-16, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1331149

RESUMO

Cottontail rabbit papillomavirus (CRPV) infection of an established cottontail epidermal cell line (Sf1Ep) resulted in the production of CRPV-specific transcripts without concomitant morphological transformation. The most abundant transcripts corresponded in size to those of the E6 and E7 open reading frames (ORFs), which are also among the commonest in domestic and cottontail rabbit papillomas. CRPV RNA production was both time- and dose-dependent, with RNA production diminishing with decreasing viral dose and increasing culture passage. Infected cultures contained episomal CRPV DNA, which did not appreciably change in abundance with time but was significantly reduced with culture passage. All features of in vitro infection, especially RNA production, were inhibited by CRPV-neutralizing, but not HPV-11-neutralizing, monoclonal antibodies. Much of this inhibition could be attributed to a blockage of viral penetration, as indicated by the reduction of CRPV DNA within virus-neutralized cultures. The results indicate that, although CRPV infection of Sf1Ep cells was abortive, it serves as a useful model for analysis of early infection events.


Assuntos
Papillomaviridae/patogenicidade , Infecções Tumorais por Vírus/etiologia , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Linhagem Celular , Transformação Celular Viral , Testes de Neutralização , Papillomaviridae/genética , Papillomaviridae/imunologia , RNA Viral/biossíntese , RNA Viral/genética , Coelhos , Transcrição Gênica
15.
J Virol Methods ; 55(2): 233-44, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8537461

RESUMO

Plasmids containing cottontail rabbit papillomavirus (CRPV) DNA can induce papillomas in vivo, but efficiency has been low. The aim of the present investigation was to explore some of the technical variables involved in inoculation of rabbits with recombinant CRPV DNA in attempts to improve both yield and consistency of papilloma induction. It was found that induction of epidermal hyperplasia, with either a mixture of turpentine and acetone or phorbol esters, produced a marked increase in papilloma yield. An additional powerful factor was the use of very vigorous, cutaneous scarification, sufficient to penetrate the papillary dermis and produce bleeding. When used in combination, papilloma yields were consistent and often reached 90-100% of inoculated sites. A number of other variables which did not consistently affect papilloma yield were tested. These included bleb and puncture injections, plasmid dose, vector type, occlusive dressings, lipofection reagent, carrier DNA, and different methods for plasmid DNA extraction and purification. It is concluded that the most important variables in improving papilloma yields were prior induction of epidermal hyperplasia and vigorous cutaneous scarification.


Assuntos
Papillomavirus de Coelho Cottontail/fisiologia , DNA Viral/metabolismo , Papiloma/virologia , Neoplasias Cutâneas/virologia , Acetona/química , Animais , Transformação Celular Neoplásica , Transformação Celular Viral , Papillomavirus de Coelho Cottontail/genética , Feminino , Hiperplasia/induzido quimicamente , Masculino , Plasmídeos , Coelhos , Terebintina
16.
Toxicology ; 136(2-3): 67-77, 1999 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-10514000

RESUMO

Toxicity studies with drugs in animals are performed to establish the toxicity profile including the no-toxic-effect-level in the animals. One of the responses used in the search for effects is organ weight. Since organ weight is related to body weight of the animal, the analysis of organ weight has to be adjusted. A traditional analysis of covariance with body weight as covariate is not always appropriate since the body weight itself can be affected by the test compound. Hence, a multivariate analysis of variance is suggested, where the correlation between organ weight and body weight is taken into account in testing an overall treatment effect. Two dimensional plots of the contour curves are used to visualize the treatment effect on organ weight and body weight simultaneously. The test procedure suggested for comparing differences between control and treated groups of animals is: start by testing hypotheses of equality of covariance matrices for the different treatment groups and then, depending on the results from the first test, test equality of the mean vectors.


Assuntos
Análise Multivariada , Tamanho do Órgão/efeitos dos fármacos , Toxicologia/estatística & dados numéricos , Algoritmos , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Ratos , Receptores de Estrogênio/agonistas , Projetos de Pesquisa
17.
Gynakol Geburtshilfliche Rundsch ; 35 Suppl 1: 25-31, 1995.
Artigo em Alemão | MEDLINE | ID: mdl-8672922

RESUMO

OBJECTIVE: A serological assay for genital HPV infection would provide important additional information to HPV DNA diagnostic methods, since it would evaluate prior exposure to the viruses, detect significant systemic immunologic response to virus infection, and could be performed in most clinical laboratories. METHODS: Serum samples from three groups of patients attending a gynecology clinic were analysed by direct ELISA for specific IgG antibodies to baculovirus-expressed HPV-6 and BPV-1-L1-VLPs. RESULTS: Positive IgG reactivity to HPV-6-L1-VLPs were 4/72 (6%) in a control group, 28/73 (38%) in a condyloma group and 17/62 (17%) in cervical intraepithelial neoplasia patients. Individual IgG ELISA values of condyloma and CIN patients for HPV-6-L1-VLPs demonstrated no correlation to results with BPV-1-L1-VLPs. CONCLUSIONS: These data show that HPV-6-L1-VLPs are effective antigens for serological studies and can detect species specific antibodies with important implications for diagnosis, epidemiology, insights to natural course of disease, prognosis and evaluation of vaccination.


Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo , Ensaio de Imunoadsorção Enzimática , Neoplasias dos Genitais Femininos/diagnóstico , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/diagnóstico , Infecções Tumorais por Vírus/diagnóstico , Adolescente , Adulto , Especificidade de Anticorpos/imunologia , Capsídeo/imunologia , Condiloma Acuminado/diagnóstico , Condiloma Acuminado/imunologia , Feminino , Neoplasias dos Genitais Femininos/imunologia , Humanos , Imunoglobulina G/sangue , Pessoa de Meia-Idade , Infecções por Papillomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Proteínas Virais
19.
Vaccine ; 24(12): 2087-93, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16343704

RESUMO

Recombinant Bacille Calmette-Guerin (rBCG) could potentially be the vaccine vehicle of choice to deliver foreign antigens from multiple pathogens. In this study we have used the cottontail rabbit papillomavirus (CRPV) rabbit model to provide a "proof of concept" that immunisation with rBCG expressing the CRPV major capsid protein, L1 (rBCG/CRPVL1), will protect outbred New Zealand White rabbits against CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were protected 5 weeks post-CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(5) cfu/ml) had papillomas, which were smaller and took longer to appear than the control rabbits. None of the negative control rabbits vaccinated with rBCG expressing an irrelevant gene or PBS were protected from CRPV challenge. Sera from rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were able to neutralise 54.5% of CRPV at serum dilutions of 1:200. These results provide evidence that BCG could potentially be used as a vaccine delivery vehicle for human papillomavirus proteins as a possible prophylactic vaccine.


Assuntos
Antígenos Virais/imunologia , Vacina BCG/administração & dosagem , Papillomavirus de Coelho Cottontail/imunologia , Infecções por Papillomavirus/prevenção & controle , Proteínas Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Vacina BCG/imunologia , Papillomavirus de Coelho Cottontail/genética , Papillomavirus de Coelho Cottontail/metabolismo , Sistemas de Liberação de Medicamentos , Imunização , Testes de Neutralização , Coelhos , Proteínas Recombinantes/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Vacinas Virais/imunologia
20.
Clin Vaccine Immunol ; 13(8): 845-53, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16893983

RESUMO

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.


Assuntos
Agrobacterium tumefaciens/genética , Antígenos Virais , Imunização , Vacinas/uso terapêutico , Proteínas Estruturais Virais , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/uso terapêutico , Sequência de Bases , Clonagem Molecular , Técnicas de Transferência de Genes , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , RNA/biossíntese , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , Vacinas/genética , Vacinas/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Proteínas Estruturais Virais/uso terapêutico
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