A rare case of massive ovarian edema in an uncomplicated singleton pregnancy.

Massive Ovarian Edema (MOE) is a rare cause of ovarian enlargement. Only 77 cases have been reported in the world literature so far, merely three of which were observed during pregnancy. Two of these showed additional pathological changes of the affected ovary (torsion) or were found in twin pregnancy. The patient presented on hand is therefore only the second report of MOE in an uncomplicated singleton pregnancy. In this case, a therapeutical resection of the affected ovary was performed at 16 + 4 weeks of gestation. The further course of the pregnancy was uncomplicated.

Tissue-specific expression of SV40 in tumors associated with the Li-Fraumeni syndrome.

Inactivation of wild-type p53 tumor suppressor function is the primary mechanism of tumor initiation in Li-Fraumeni syndrome (LFS) individuals with germline p53 mutations. Tumors derived from LFS patients frequently retain the normal p53 allele, suggesting that alternative mechanisms in addition to gene deletion must be involved in inactivating wild-type p53 protein. DNA tumor viruses, such as SV40, target p53 for inactivation through the action of viral oncoproteins. We studied the probands from two unrelated LFS families, each of whom presented with multiple malignant neoplasms. Patient 1 developed an embryonal rhabdomyosarcoma (RMS) and a choroid plexus carcinoma (CPC), while patient 2 developed a CPC and subsequently presented with both an osteosarcoma (OS) and renal cell carcinoma (RCC). We utilized DNA sequence analysis and immunohistochemistry to determine p53 gene status in the germline and tumors, as well as evidence for SV40 T-antigen oncoprotein expression. Each patient harbored a heterozygous germline p53 mutation at codons 175 and 273, respectively. In patient 1, the normal p53 gene was lost while the mutant p53 allele was reduced to homozygosity in the RMS. Both normal and mutant genes were maintained in the CPC. In patient 2, normal and mutant p53 alleles were retained in both the CPC and RCC. Both specific PCR and immunostaining detected SV40 T-antigen in both CPCs and the RCC. In addition to chromosomal alterations, epigenetic mechanisms may disrupt p53 function during tumorigenesis. In two LFS patients, we found SV40 DNA sequences and viral T-antigen expression that could account for inactivation of the normal p53 protein. Inactivation of p53 or other tumor suppressors by viral proteins may contribute to tumor formation in specific tissues of genetically susceptible individuals.

Immunization with a pentameric L1 fusion protein protects against papillomavirus infection.

The prophylactic papillomavirus vaccines currently in clinical trials are composed of viral L1 capsid protein that is synthesized in eukaryotic expression systems and purified in the form of virus-like particles (VLPs). To evaluate whether VLPs are necessary for effective vaccination, we expressed the L1 protein as a glutathione S-transferase (GST) fusion protein in Escherichia coli and assayed its immunogenic activity in an established canine oral papillomavirus (COPV) model that previously validated the efficacy of VLP vaccines. The GST-COPV L1 fusion protein formed pentamers, but these capsomere-like structures did not assemble into VLPs. Despite the lack of VLP formation, the GST-COPV L1 protein retained its native conformation as determined by reactivity with conformation-specific anti-COPV antibodies. Most importantly, the GST-COPV L1 pentamers completely protected dogs from high-dose viral infection of their oral mucosa. L1 fusion proteins expressed in bacteria represent an economical alternative to VLPs as a human papillomavirus vaccine.

Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1.

The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.

Antiviral T-cell-independent type 2 antibody responses induced in vivo in the absence of T and NK cells.

Polyomavirus (PyV) infection induces protective T-cell-independent (TI) IgM and IgG responses in T-cell-deficient (TCR beta x delta-/-) mice. In this study, we show that PyV is a TI -2 antigen: B cells with a mutated Bruton's tyrosine kinase (Xid mutants) do not respond to PyV with antibody secretion in the absence of T cells. We also demonstrate that NK-cell-mediated "help" is not absolutely required for the induction of the TI-2 antibodies to PyV; thus for the first time, we provide evidence for protective IgM and IgG responses against a viral infection induced in mice lacking T and NK cells (CD3Etg). Comparison of the antibody responses observed in T- and NK-cell-deficient mice with those of mice lacking only T cells, however, suggests that NK cells may promote isotype switching to IgG2a. This effect is probably mediated by IFN gamma secretion. In support of this idea, studies on the antibody responses of PyV-infected SCID mice that had been reconstituted with IFN gamma R-/- B cells or wild-type B cells demonstrated the IFN gamma dependence of PyV-specific TI IgG2a secretion and provided evidence that IFN gamma acting directly on B cells plays an important role in TI pathways of isotype switching to IgG2a in vivo.

SV40: A human pathogen?

Over the past eight years an increasing number of investigators have found SV40 genomic sequences in a variety of human samples, both malignant and normal. Tumor types recurrently reported as SV40-positive include choroid plexus neoplasms, ependymomas, osteosarcomas, and mesotheliomas. Nonetheless, considerable skepticism that SV40 is a human pathogen still prevails. More constructively, the study of SV40 in humans has renewed interest in the related BK and JC viruses and their role in human disease. New questions now must be addressed. In particular, seroepidemiologic studies utilizing reagents that distinguish SV40, BKV, and JCV immune responses would be a logical next step for independently assessing viral prevalence. Also, prospective studies of select patient groups using optimized detection methods might determine whether SV40 is associated with human oncogenesis in particular circumstances. The importance of such research is underscored by the potential to prevent human polyomavirus infections, and possible associated malignancy, through immunization of high risk populations.

Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16.

The papillomavirus major late protein, L1, forms the pentameric assembly unit of the viral shell. Recombinant HPV16 L1 pentamers assemble in vitro into capsid-like structures, and truncation of ten N-terminal residues leads to a homogeneous preparation of 12-pentamer, icosahedral particles. X-ray crystallographic analysis of these particles at 3.5 A resolution shows that L1 closely resembles VP1 from polyomaviruses. Surface loops contain the sites of sequence variation among HPV types and the locations of dominant neutralizing epitopes. The ease with which small virus-like particles may be obtained from L1 expressed in E. coli makes them attractive candidate components of a papillomavirus vaccine. Their crystal structure also provides a starting point for future vaccine design.

The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses.

Polyomavirus (PyV) infection elicits protective T cell-independent (TI) IgG responses in T cell-deficient mice. The question addressed in this report is whether CD40 signaling plays a role in this TI antiviral IgG response. Because CD40 ligand (CD40L) can be expressed on numerous cell types in addition to activated T cells, it is possible that cells other than T cells provide CD40L to signal through CD40 on B cells and hence positively influence the antiviral TI IgG responses. In this study we show, by blocking CD40-CD40L interactions in vivo with anti-CD40L Ab treatment in TCR betaxdelta-/- mice and by using SCID mice reconstituted with CD40-/- B cells, that the lack of CD40 signaling in B cells results in a 50% decrease in TI IgG secreted in response to PyV. SCID mice reconstituted with CD40L-/- B cells also responded to PyV infection with diminished IgG secretion compared with that of SCID mice reconstituted with wild-type B cells. This finding suggests that B cells may provide the CD40L for CD40 signaling in the absence of T cell help during acute virus infection. Our studies demonstrate that, although about half of the TI IgG responses to PyV are independent of CD40-CD40L interactions, these interactions occur in T cell-deficient mice and enhance antiviral TI Ab responses.

"Local rules" theory applied to polyomavirus polymorphic capsid assemblies.

The papovaviruses are nonenveloped dsDNA viruses whose capsids are characterized by a non-quasi-equivalent bonding pattern in which 72 pentameric capsomeres occupy positions having either five or six neighboring capsomeres. The local rules theory of Berger et al. (1994, Proc. Natl. Acad. Sci. USA, 91, 7732-7736), previously developed to explain aspects of icosahedral capsid assembly, has been applied to the papovavirus geometry. Local rules describe capsid symmetry patterns in terms of the local interactions of assembly units, such as coat proteins or capsomeres. Polymorphic assemblies, including T = 1 icosahedral, dodecahedral, spiral, and tubular structures of the polyomavirus VP1 protein, can be induced by specific mutations or changes in the solvent conditions during in vitro assembly of the recombinant coat protein. Local rules models were developed to model the wild-type capsid and several polymorphic assemblies. Some assemblies corresponded to structures modeled by small deviations from wild-type local rules. We conclude that aspects of polyomavirus assembly are consistent with local rules models, although they do not explain all polymorphisms. These results may provide insights into the nature of papovavirus assembly, constraints on assembly pathways, and strategies for disrupting assembly.

[The ICSI (Intracytoplasmic Sperm Injection)]. / La ICSI (Intracytoplasmic Sperm Injection).

ICSI (Intracytoplasmic Sperm Injection) is the latest known assisted reproduction technique (ART) and it already appears to be mature. In fact the analysis of the results presented by the researchers over the years has shown that the most specific indication for this ART is the sterility of the couple with serious male pathology, up to ejaculatory azoospermia where it is possible to perform MESA, PESA or TESE. Any kind of sterility could be solved with ICSI whose only limit presently known is the high technology and therefore high costs involved. The percentage of oocytes that undergo ICSI without being damaged varies from 87% to 94% and the percentage of fertilization varies from 33% to 71%. The transfer rate is 59-100%. The rate of pregnancies per couple ranges from 12% to 40% of the couples, from 11% to 41% for the transfers. Since abortions are similar to the values of the normal population (10-15%), ICSI is actually the assisted fertilization technique with the highest incidence of pregnancies and "take home babies". The percentage incidence of the two sexes, of the malformations and the typologies of malformations corresponds to those observed in the population with spontaneous pregnancies. Since there is no natural selection of the gametes in ICSI, one may be sure that when spermatozoa with any kind of pathology are injected, the pregnancy does not take place at all.

Simian virus 40 DNA sequences in human brain and bone tumours.

This report reviews recent observations regarding the association of simian virus 40 (SV40) DNA sequences with brain and bone tumours of childhood [1-3]. Our initial investigation was suggested by the tumorigenicity of SV40 in animals, and the transgenic mouse expression of SV40 large T-antigen in which all animals developed choroid plexus (CP) tumours. Polymerase chain reaction (PCR) analysis and DNA sequencing demonstrated SV40-like DNA sequences, amplified from the "Rb-pocket" binding domain of the viral large T-antigen, in 10/20 CP and 10/11 ependymoma tumours of children. The PCR analysis was subsequently extended to three additional regions of the viral genome: the carboxy-terminal region of large T-antigen, the viral enhancer/origin, and the VP1 gene. All amplified products were related to SV40 sequences. Furthermore, because one individual in the original brain tumour study was a member of a Li-Fraumeni kindred, 151 DNA samples from such families were analysed. Only 18 were positive for viral sequences and 11 of these were isolated from individuals with osteosarcomas. This observation led to a further analysis of DNA from bone tumours, in which 54/160 samples contained SV40-like sequences. These studies associate SV40-like sequences with human CP, ependymoma, and bone tumours. A causal relationship to human oncogenesis remains a subject for further study.

Spontaneous pregnancy after 13 years of amenorrhea in a patient with a voluminous ovarian dysgerminoma and submitted to left adnexectomy and radiotherapy.

The authors illustrate the case of a 17-year-old patient who was submitted to left adnexectomy in view of an ovarian dysgerminoma 24 cm in diameter and weighing 2,800 g. She was subsequently submitted to two cycles of radiotherapy. Following a period of amenorrhea lasting 13 years and characterized by high serum levels of gonadotropins, the patient had a spontaneous pregnancy and at 33 weeks of gestation delivered a live and vital fetus. Therefore the occurrence of post-radiotherapy amenorrhea, characterized by high serum gonadotropin levels, should not always be considered pathognomonic of precocious menopause. The possibility that radiotherapy causes only a temporary alteration in ovarian activity should also be taken into consideration.

T-Cell-independent immunoglobulin G responses in vivo are elicited by live-virus infection but not by immunization with viral proteins or virus-like particles.

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor beta chain [TCR-beta] -/- or TCR-betaxdelta -/-) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.

Human papillomavirus type 11 recombinant L1 capsomeres induce virus-neutralizing antibodies.

The human papillomavirus type 11 (HPV-11) L1 major capsid protein can be trypsinized to generate recombinant capsomeres that retain HPV genotype-restricted capsid antigenicity (M. Li, T. P. Cripe, P. A. Estes, M. K. Lyon, R. C. Rose, and R. L. Garcea, J. Virol. 71:2988-2995, 1997). In the present study, HPV-11 virion-neutralizing monoclonal antibodies H11.F1 and H11.H3, previously characterized as recognizing two distinct HPV-11 capsid-neutralizing antigenic domains (S. W. Ludmerer, D. Benincasa, and G. E. Mark III, J. Virol. 70:4791-4794, 1996), were each found to be highly immunoreactive with trypsin-generated capsomeres in an enzyme-linked immunosorbent assay (ELISA). Capsomeres were used to generate high-titer polyclonal immune sera that demonstrated HPV genotype-restricted reactivity by ELISA. The capsomere antisera were then tested in an in vitro infectivity assay and found to neutralize HPV-11 virions. In this assay, HPV-11 capsomere polyclonal antisera exhibited neutralization titers (10(-5) to 10(-6)) comparable to those obtained with a virion-neutralizing antiserum raised previously against intact HPV-11 VLPs (R. C. Rose, R. C. Reichman, and W. Bonnez, J. Gen. Virol. 75:2075-2079, 1994). These results indicate that highly immunogenic, genotype-restricted HPV capsid-neutralizing antigenic domains are contained entirely within capsomeres. Thus, capsomeres may be viable vaccine candidates for the prevention of HPV disease.

Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly.

In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions. At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy. However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I. When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion. Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value. A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly. A recombinant HPV type 11 L1 protein, which was purified after expression in Escherichia coli and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures. These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm.

Expression of the human papillomavirus type 11 L1 capsid protein in Escherichia coli: characterization of protein domains involved in DNA binding and capsid assembly.

The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to assemble further. In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells. The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein. The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.