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F Breitburd

Publications and source records attributed to F Breitburd.

At least 19 recordsLinked to original sources
PubMed2000

Variation in the nucleotide sequence of cottontail rabbit papillomavirus a and b subtypes affects wart regression and malignant transformation and level of viral replication in domestic rabbits.

We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides. The most variable region (7% nucleotide divergence) included the long regulatory region (LRR) and the E6 and E7 genes. A mutation in the stop codon resulted in an 8-amino-acid-longer CRPVb E4 protein, and a nucleotide deletion reduced the coding capacity of the E5 gene from 101 to 25 amino acids. In domestic rabbits homozygous for a specific haplotype of the DRA and DQA genes of the major histocompatibility complex, warts induced by CRPVb DNA or a chimeric genome containing the CRPVb LRR/E6/E7 region showed an early regression, whereas warts induced by CRPVa4 or a chimeric genome containing the CRPVa4 LRR/E6/E7 region persisted and evolved into carcinomas. In contrast, most CRPVa, CRPVb, and chimeric CRPV DNA-induced warts showed no early regression in rabbits homozygous for another DRA-DQA haplotype. Little, if any, viral replication is usually observed in domestic rabbit warts. When warts induced by CRPVa and CRPVb virions and DNA were compared, the number of cells positive for viral DNA or capsid antigens was found to be greater by 1 order of magnitude for specimens induced by CRPVb. Thus, both sequence variation in the LRR/E6/E7 region and the genetic constitution of the host influence the expression of the oncogenic potential of CRPV. Furthermore, intratype variation may overcome to some extent the host restriction of CRPV replication in domestic rabbits.

Animals↗
PubMed1999

Human papillomavirus vaccines.

Genital human papillomavirus (HPV) infections are the viral sexually transmitted diseases most frequently diagnosed that include anogenital condylomas and squamous intra-$bepithelial lesions, among which the precursors of invasive carcinomas of the uterine cervix. In animal PV models, vaccination against L1 and/or L2 viral capsid proteins provides an efficient protection against infection, involving virus type-specific neutralizing antibodies. Vaccination against non-structural E1, E2, E6 or E7 viral proteins does not prevent infection, unless administered altogether, but tends to stimulate regression, warranting the design of therapeutic vaccines. Prophylactic vaccines based on the use of virus-like particles (VLPs) obtained by auto-assembly of L1 or L1 and L2 proteins produced by recombinant DNA technology are under phase I/II clinical trials for HPV6/11 associated with condylomas and for HPV16, the most frequent oncogenic genotype. Second generation vaccines are chimeric proteins or VLPs incorporating one of the structural proteins (L1 or L2) fused to a non-structural protein (E6, E7 or E2), which should induce both humoral and cellular immunity. Vaccine valency (number of genotypes), route of administration (humoral versus local immunity), vaccinees (children, young adults, gender) and forms of vaccines (recombinant $LSalmonella typhimurium*I$L, edible plants expressing L1 and L2 proteins, DNA vaccines, synthetic antigenic peptides) are under study. End points to evaluate vaccine efficacy in phase III trials should include viral DNA detection and typing, and screening for low or high grade intraepithelial lesions. Therapeutic vaccines based on recombinant HPV E6 and/or E7 vaccinia virus, L2-E7 fusion proteins or E7 peptides corresponding to cytotoxic T cell epitopes are currently tested (phase I/II trials) in patients with cervical carcinomas of advanced clinical stages or high grade intraepithelial lesions. Animal studies, phase I/II clinical trials and implementation of the community support that HPV vaccines will constitute an efficient means to prevent carcinoma of the uterine cervix.

Animals↗
PubMed1997

A cottontail rabbit papillomavirus strain (CRPVb) with strikingly divergent E6 and E7 oncoproteins: An insight in the evolution of papillomaviruses.

We previously observed that warts induced by an isolate of cottontail rabbit papillomavirus (CRPV) showed incomplete instead of systemic regression in some domestic rabbits. We report that the viral isolate contained, as a major component, a CRPV strain (CRPVb) showing an unexpectedly high divergence in the E6 and E7 open reading frames (ORFs), compared to the prototype CRPVa present in the isolate as a minor component. The E6 and E7 oncoproteins of CRPVa and -b disclosed only 87.5% identical amino acids and differed in size by three and two amino acids, respectively. This divergence involved (i) a great number (4.4%) of nucleotide substitutions and a high rate (83.3%) of nonsynonymous mutations; (ii) mutations changing the E6 and E7 stop codons; and (iii) in-frame sequence insertions in the E6 ORF (18 nucleotides) and downstream of the mutated E7 stop codon (6 nucleotides), both likely to result from a duplication of adjacent sequences. These extensive differences could account for distinct biological and antigenic properties. Strikingly, only four (0.8%) amino acids of the L1 major capsid protein were variable. Thus, it seems likely that sequence duplications and mutations affecting stop codons exert a strong selection pressure on the fixation of nonsynonymous mutations and that phylogenetic calculations based only on point mutations may misevaluate the time scale of the evolution of papillomaviruses.

Amino Acid Sequence↗
PubMed1996

HLA control in the progression of human papillomavirus infections.

Cellular immunity is likely to be of major importance for the clearance of inapparent or overt infections caused by human papillomaviruses (HPVs). The highly polymorphic class I or class II human leukocyte antigen (HLA) molecules present HPV-derived peptides to cytotoxic (CD8+) or helper (CD4+) T lymphocytes bearing specific receptors and condition the immune responsiveness to HPV infections. Recent data point to a role of an altered expression of HLA molecules in the persistence of HPV-induced cervical premalignant lesions and their progression towards invasive carcinoma. Furthermore positive of negative associations of certain HLA alleles or haplotypes with cutaneous of cervical neoplasias have been found. These observations may have important implications in the design of therapy or vaccines aimed at eradicating cervical cancer.

Animals↗
PubMed1996

Progressive growth of human papillomavirus type 16-transformed keratinocytes is associated with an increased release of soluble tumour necrosis factor (TNF) receptor.

Analysis of conditioned media generated by weakly and highly tumorigenic SKv-1 keratinocyte lines harbouring integrated human papillomavirus type 16 (HPV16) DNA sequences revealed a factor inhibiting TNF-alpha and TNF-beta cytotoxic activity. This inhibitory activity was specifically blocked by htr-9 monoclonal antibody (MAb) recognising 55/60 kDa type I TNF receptor suggesting that it is related to a soluble form of this particular receptor (sTNF-RI). The presence of sTNF-RI was confirmed by Western blot analysis of SKv-1 cell-conditioned medium showing a band of 31.5 kDa as well as by the specific enzyme-linked immunobiological assay (ELIBA). Release of sTNF-RI was a result of shedding because Northern blot analysis showed that SKv-1 cells expressed a full-length TNF-RI mRNA, and radioimmunoprecipitation of TNF-RI from [32S]cysteine-labelled cell extracts demonstrated the presence of normal 55 kDa molecule. Evaluation by ELIBA showed that highly tumorigenic SKv-12 cells released significantly more sTNF-RI than their weakly tumorigenic SKv-11 parental cells. Furthermore, human recombinant as well as SKv cell-derived sTNF-RI stimulated proliferation of weakly tumorigenic SKv-11 cells. This suggests that a progressive growth of some neoplastic cells may be, at least partially, a result of an increased spontaneous release of sTNF-RI that enables the cells to escape from local TNF-alpha-mediated growth inhibition.

Carcinoma in Situ↗
PubMed1995

Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection.

We tested the ability of vaccination with virus-like particles (VLPs) to protect domestic rabbits against papillomas induced by the cottontail rabbit papillomavirus (CRPV). A recombinant baculovirus system that expressed only the L1 major papillomavirus structural protein or L1 plus the minor L2 protein was used in insect cells as the source of VLPs. Groups of 10 rabbits were immunized with native or denatured VLPs from CRPV or type 1 bovine papillomavirus by using Freund's adjuvant. Alum was used as the adjuvant for an additional group immunized with CRPV L1-L2 VLPs. Animals were challenged with 5 x 10(10) and 2 x 10(11) particles on opposing flanks. No protection was seen in rabbits immunized with native or denatured bovine papillomavirus L1-L2 or with denatured CRPV L1-L2. In these groups, the lower and higher challenge doses resulted in 27 of 30 animals with extensive papillomas, with each of the remaining animals having a smaller number of persistent papillomas. Progression to carcinoma developed in 20 rabbits. Animals inoculated with native CRPV VLPs composed of L1 alone or L1-L2 developed many fewer lesions; the lower and higher challenge doses resulted in 17 of 29 and 5 of 29 rabbits, respectively, with no lesions, and the remainder developed only one to eight papillomas, which all regressed except for those on 1 rabbit. None developed cancer within 1 year of infection. Rabbits vaccinated with native CRPV VLPs developed high-titer antibodies in an enzyme-linked immunosorbent assay based on native VLPs, and passive transfer of serum or immunoglobulin G from rabbits immunized with CRPV VLPs protected against CRPV challenge. We conclude that native VLPs can induce antibody-mediated, type-specific protection against experimental papillomavirus infection.

Animals↗
PubMed1995

Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor.

Virus-like particles (VLPs) composed of L1 derived from bovine papillomavirus type 1 (BPV-1), several human papillomavirus types, or cottontail rabbit papillomavirus (CRPV) agglutinated mouse but not human or rat erythrocytes. Treatment of mouse erythrocytes with trypsin prevented hemagglutination (HA) by BPV-1. Sera from rabbits immunized with native CRPV VLPs, which protect against experimental CRPV infection, exhibited high titers of antibodies that inhibited CRPV VLP HA activity, while sera from rabbits immunized with denatured CRPV VLPs or native BPV VLPs, which do not protect against CRPV infection, were not inhibitory. Testing for HA inhibition is a rapid and simple method for examining the serological relatedness of papillomaviruses and measuring protective antibody titers after VLP vaccination.

Animals↗
PubMed1994

A mouse model for studying epidermodysplasia-verruciformis-associated carcinogenesis.

Epidermodysplasia verruciformis (EV) is considered as a model of genetic cancer due to unusual susceptibility to EV-specific human papillomaviruses (HPVs). We established an in vivo experimental system for long-term propagation of EV tissue which should facilitate the study of tumor progression in EV. Skin fragments from benign and early pre-malignant lesions of 6 EV patients were grafted under the kidney capsule of athymic mice. After several months the grafted tissue formed epidermal cysts. These cysts showed typical EV cytopathic effect, in situ hybridization revealed the presence of HPV 5, 8, 9, 12 and 36 DNA, and immunoperoxidase staining detected papillomavirus-group-specific antigens in the permissive cells of the cysts. In some cysts, there were numerous mitoses and downward proliferation of the epidermis with slight dyskeratotic changes. The experimental system described constitutes a model for studies on the role of HPV and other co-factors in human cutaneous carcinogenesis.

Animals↗
PubMed1994

Increased tumorigenicity of human keratinocytes harboring human papillomavirus type 16 is associated with resistance to endogenous tumor necrosis factor-alpha-mediated growth limitation.

The aim of this study was to evaluate the relationship between tumorigenicity of cell sublines derived from weakly tumorigenic SKv-e and SKv-l keratinocytes harboring human papillomavirus type 16 (HPV16) and their susceptibility to autocrine growth limitation mediated by tumor necrosis factor-alpha (TNF-alpha). These sublines displayed different in vitro proliferative potential which correlated with tumorigenicity in nu/nu mice. Recombinant TNF-alpha inhibited in vitro growth of weakly tumorigenic parental SKv cell lines while it did not affect proliferation of their respective highly tumorigenic sublines. Resistance to TNF-alpha correlated with both increased in vitro proliferation and tumorigenicity. Anti-TNF-alpha antibodies (Ab) significantly increased in vitro proliferation of weakly tumorigenic parental SKv cells up to the levels of their highly tumorigenic sublines. Growth of highly tumorigenic SKv cells was not affected. On the other hand, proliferation of SKv cells was affected neither by transforming growth factor-beta (TGF-beta) nor by anti-TGF-beta Ab. All SKv cell sublines tested spontaneously released TNF-alpha, as evaluated by a specific radioimmunoassay; however, the levels of the endogenous cytokine were not related to their proliferative potential and tumorigenicity. An increased resistance to the anti-proliferative effect of TNF-alpha may be associated with decreased expression of TNF-alpha receptors (TNF-alpha R) inasmuch as evaluation of 125I-TNF-alpha binding and Northern-blot analysis of TNF-alpha R-specific mRNA showed that highly tumorigenic SKv cell sublines expressed significantly lower numbers of TNF-alpha R than their respective parental cells. These results show that an increased tumorigenicity of HPV16-harboring SKv keratinocytes may be, at least partially, due to escape from autocrine TNF-alpha-mediated growth limitation.

Animals↗
PubMed1994

Analysis of the nucleotide sequence variation of the antigen-binding domain of DR alpha and DQ alpha molecules as related to the evolution of papillomavirus-induced warts in rabbits.

We previously found that regression of skin warts induced by the Shope cottontail rabbit papillomavirus in New Zealand White rabbits, as well as malignant conversion of persistent warts, are linked to a restriction fragment length polymorphism of the major histocompatibility complex class II DR alpha and DQ alpha genes. To find out whether this immunogenetic control could be connected with the antigen binding and presentation function of the alpha 1 domain of class II molecules, we have sequenced the exon 2 of the four DR alpha EcoRI and six of the seven DQ alpha PvuII restriction fragment length polymorphism alleles identified, and deduced the encoded amino acid sequences. We found no amino acid polymorphism among DR alpha alleles, indicating that the alpha 1 domain of the DR alpha chain does not condition wart regression or cancer development. In contrast, 27 of the 82 amino acids of the DQ alpha 1 domain were found variable, defining five amino acid sequence alleles. The restriction fragment length polymorphism allele linked to regression and another allele not linked to regression share the same alpha 1 domain, indicating that wart regression is rather conditioned by a closely linked gene. The most divergent DQ alpha 1 allele, however, was that associated with a higher risk of cancer. Alignment of rabbit and human DQ alpha exon 2 alleles disclosed that amino acid charge variations occur at positions assumed to be important for peptide binding in humans. By modulating the affinity for tumor-specific antigenic peptides, such transitions could affect immune surveillance and, thus, condition the risk for progression to carcinoma of papillomavirus-associated lesions.

Alleles↗
PubMed1993

Cytopathic effect in human papillomavirus type 1-induced inclusion warts: in vitro analysis of the contribution of two forms of the viral E4 protein.

Myrmecia warts induced by human papillomavirus type 1 (HPV1) are characterized by abundant eosinophilic inclusions associated with HPV1 E4 gene products. The major HPV1 E4 proteins are a 17-kilodalton (kDa) E1-E4 fusion protein and a 16-kDa species lacking the five E1 amino acids and a few E4 residues. To study the contribution of E4 proteins to the formation of myrmecia inclusions, we used a previously designed transient expression system in the rabbit VX2-R keratinocyte line. We find that the E1-E4 and an E4 protein without the E1 residues (E4-3200) form eosinophilic inclusions. Ultrastructural and immunoelectron microscopic studies show that the electron-dense, keratohyalin-like myrmecia inclusions are recognized by anti-E4 antibodies. They are associated with tonofilament bundles at their periphery in the cytoplasm or free of filaments in the nucleus. The E1-E4 inclusions formed in vitro are also homogeneously electron dense, and are usually associated with tonofilaments at their periphery in the cytoplasm and free of filaments in the nucleus. The E4-3200 inclusions are exclusively cytoplasmic and heterogeneously electron dense, with a fibrillar structure made of entangled 10-nm filaments. The expression of either protein in VX2-R cells does not result in the collapse of the cytokeratin network, as shown by immunofluorescence double-labeling experiments. This is in contrast to data reported for the HPV16 E1-E4 protein. Our findings indicate that the E1-E4 protein by itself accounts for the formation of myrmecia inclusions, and suggest that the five N-terminal E1 amino acids play a major role in the interaction of E4 proteins with intermediate filaments.

Adenovirus E4 Proteins↗
PubMed1992

Linkage of regression and malignant conversion of rabbit viral papillomas to MHC class II genes.

Human papillomaviruses associated with cutaneous and anogenital cancers induce intraepithelial precursor lesions which may regress spontaneously or progress into invasive carcinomas. Cell-mediated immune responses are probably involved in regression of precancerous lesions and the polymorphism of the genes responsible may thus have a key role in the variability of the host response. Skin warts and cancers induced in rabbits by Shope papillomavirus provide a model to test this hypothesis. We analysed a restriction-fragment-length polymorphism of major histocompatibility complex class I and class II genes and T-cell receptor beta-chain genes in infected domestic rabbits. We found a strong linkage between wart regression and a DR alpha EcoRI fragment, and an increased relative risk of malignant transformation associated with a DQ alpha PvuII fragment. This indicates a genetic control of wart evolution, involving genes in the class II region of the major histocompatibility complex.

Animals↗
PubMed1992

Human papillomavirus type 1 E4 proteins differing by their N-terminal ends have distinct cellular localizations when transiently expressed in vitro.

Two major human papillomavirus type 1 (HPV-1) E4 proteins are found in large amounts in productively infected differentiating wart cells, a 17-kDa protein translated from an E1-E4 transcript and a processed 16-kDa protein lacking the E1 amino acids at least. The functions of the E4 proteins are still unknown. We have designed an in vitro system allowing the transient expression of three forms of HPV-1 E4 proteins: the 17-kDa E1-E4 protein, an E4 protein without the five E1 amino acids (E4-3200), and E4 protein initiated at the E4 ATG located upstream of the splice acceptor site (E4-3181). The E4-3181 protein has five additional N-terminal amino acids compared with E4-3200. The E4-3181 protein has not yet been detected in vivo but could, in principle, be translated from any transcript encoding the E2 protein. The constructs were transfected in two keratinocyte lines, one derived from a rabbit transplantable carcinoma (VX2R) and the other from a human penile carcinoma (SK-p). E4 transcripts with the expected size were detected in both cell lines by Northern (RNA) blot analysis. Surprisingly, the E4 proteins were found only in the VX2R cells by radioimmunoprecipitation and immunofluorescence experiments. The E1-E4 and the E4-3200 proteins were both cytoplasmic and were associated with granules reminiscent of the cytoplasmic inclusions pathognomonic of the HPV-1 infection. Moreover, each protein showed a specific staining pattern of the inclusions. In contrast, the E4-3181 protein was essentially intranuclear and perinuclear. Thus, HPV-1 E4 proteins differing in their N-terminal ends have distinct cellular localizations and arrangements. It is tempting to assume that this may relate to different roles.

Base Sequence↗
PubMed1990

Expression of the human papillomavirus type 16 genome in SK-v cells, a line derived from a vulvar intraepithelial neoplasia.

The SK-v cells, established from a premalignant vulvar lesion, contain human papillomavirus type 16 (HPV-16) sequences integrated at a single cellular site and derive from a cell clone present in vivo. Transcription of the HPV-16 genome in SK-v cells was analysed by cDNA heteroduplex mapping and sequencing, and by RNase mapping. Viral sequences were shown to be transcribed into virus-cell fusion messengers. The two major transcripts have a coding capacity for a truncated E6 protein, an E7 protein and an E1-E4 fusion protein, but differ in their 3' virus-cell junction. Minor transcripts have a coding capacity for a full-length E6 protein and another truncated version of E6. The transcription pattern in the E6-E7 region was found to be the same both in SK-v cells and in CaSki cells, a line derived from an invasive cervical carcinoma. Immunoprecipitation experiments showed that the E6 protein (18K) and, predominantly, the E7 protein (20K) are expressed in SK-v cells as in CaSki cells. The E7 protein was found in a two- to threefold lower amount in SK-v cells, but showing the same half-life (about 1 h).

Base Sequence↗
PubMed1986

A new method for studying epidermalization in vitro.

A new method for studying epidermalization in vitro is described. It consists of inserting a punch biopsy that serves as a source of epidermis into dermal equivalent freshly made up, with fibroblasts mixed in a collagen matrix. Fibroblasts cling to collagen fibrils and contract the matrix, leading in 3 days to a resistant dermal equivalent holding the punch biopsy firmly in place. At day 5, a culture medium favouring epidermal growth was used and a fringe of a new epidermis appeared around the punch, the area of which grew linearly with time. This new epidermis showed a pattern of differentiation similar to epidermis in vivo, with cuboidal basal cells, keratohyalin granules, membrane coating granules and the expression of the 65-67 kd keratin subset. The method seems to combine the advantages of the explant technique and of classical keratinocyte cultures, providing the researcher with a large quantity of differentiated epidermis, the pharmacologist with simple and quantitative system in which to study modifications of growth and differentiation of epidermis, and the plastic surgeon with a possible material for skin grafting.

Biopsy↗
PubMed1986

The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.

The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions. Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.

Antigens, Viral↗