Expression of Epstein-Barr virus lytically related genes in African Burkitt's lymphoma: correlation with patient response to therapy.

A study on the Epstein-Barr virus (EBV)-associated malignancy (endemic) Burkitt's lymphoma (BL) was initiated on fine-needle-aspiration biopsies from 46 proven BL cases in Malawi. Gene expression that might correlate with patient serology (where high levels of antibodies to lytically related genes are commonly observed) was explored. In two-thirds of the cases, we identified the EBV BZLF1 replication activator intermediate early protein ZEBRA in varying quantities and to varying extents in cells by immuno-cytochemistry. The early lytic-cycle gene transcript BHLF1 was assessed positively by solid-phase hybridisation in over half of the same tumours. Evidence of transcription of these genes was confirmed on a smaller number of surgically removed fresh biopsies by RT-PCR. We asked whether our findings, which are generally counter to the established notion that EBV gene expression in BLs is restricted to the latent function, EBNA1, might offer some explanation for the differential responses to chemotherapy observed among African patients. Where the duration of follow-up was sufficient to assign the cases (37 in number) to one of 3 categories, namely, complete, partial or no response, a significant correlation between expression of the viral function ZEBRA and a positive patient response to treatment was found. Lack of this was associated with poor prognosis. Clinical data and EBV gene expression results support the postulate of subgroups of African BLs, the intermediate early antigen providing a marker of potential use in patient management.

Immunization of common marmosets with Epstein-Barr virus (EBV) envelope glycoprotein gp340: effect on viral shedding following EBV challenge.

Epstein-Barr virus (EBV), the cause of infectious mononucleosis, is involved in the pathogenesis of several human cancers, the highest frequency of association being found in undifferentiated nasopharyngeal carcinoma and endemic Burkitt's lymphoma. The development of animal models in which potential vaccines can be tested is important. EBV infection of the common marmoset, using the M81 strain originally derived from a patient with nasopharyngeal carcinoma, induces a carrier state in this animal. Persistent infection is characterized by the production of antibodies to viral antigens, and the secretion of EBV DNA into buccal fluids. Following immunization with envelope glycoprotein gp340 derived from a bovine papilloma virus expression vector, prior to EBV infection, viral DNA was detected significantly less frequently in the buccal fluids of immunized, than of nonimmunized, infected animals, indicating that although the carrier state had not been abolished, it had been altered. A reduction in virus load was also observed when offspring of seronegative, and on occasion seropositive, parents were immunized neonatally, before EBV challenge.

Direct demonstration of persistent Epstein-Barr virus gene expression in peripheral blood of infected common marmosets and analysis of virus-infected tissues in vivo.

Epstein-Barr virus (EBV) infection in animal model systems has been studied previously in marmosets and tamarins using serology and PCR of saliva. Here we directly demonstrated long-term persistence of EBV in the peripheral blood of marmosets by assaying EBER RNA expression. A new reverse transcription-PCR assay, able to distinguish a naturally occurring strain polymorphism in EBER 2 that may be useful as a strain marker for monitoring persistence and interactions between multiple strains in the same animal or person, has been developed. In situ hybridization and immunohistochemistry have also been used to search for EBV-infected cells in the animals. The carrier state in the common marmoset is similar to that of humans in that it is asymptomatic, long-lived and displays a very low level of circulating virus-infected cells. It differs from the human in lacking the characteristic antibody response to EBNA 1.

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life-threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95-8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton-top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild-type strains of EBV in the general population than does the standard B95-8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95-8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

Persistent Epstein-Barr virus infection in the common marmoset (Callithrix jacchus).

Epstein-Barr virus (EBV) infection of the common marmoset causes long-term infection, with production of antibodies to virus-induced antigens, without clinical illness. Attempts to show the presence of EBV DNA in saliva of infected animals by PCR were initially unsuccessful, although slot-blot hybridization analysis demonstrated that viral DNA was present. Further investigations showed that most samples of pilocarpine-induced saliva, and 33% of the samples of whole mouth fluids (WMF) tested, were inhibitory to PCR. Similar results were found using human WMF. A method of assessing samples of marmoset WMF for the presence of EBV, by PCR using an EBV BamHI W probe, and removing inhibition with Chelex 100, is described. A total of 202 samples from 21 EBV infected, and seven non-infected animals was tested. Five seropositive animals shed virus on every occasion, and 15 intermittently. Two marmosets, infected as neonates, showed progressively increasing humoral responses to viral antigens, and shed virus on every occasion tested over 3 years. When mated with uninfected animals, the latter seroconverted 4 and 6 weeks later, respectively, and later shed virus into their WMF. The naturally infected animals were paired with naive marmosets, and were able to pass on infection. These results establish that long-term, permissive EBV infection occurs in the common marmoset, and demonstrate again the similarities in the response to EBV between marmoset and man.

Analysis of the tumorigenic potential of common marmoset lymphoblastoid cells expressing a constitutively activated c-myc gene.

The respective roles of Epstein-Barr virus (EBV) and c-myc in the pathogenesis of endemic Burkitt's lymphoma (BL) are unclear. In order to help resolve the question whether constitutive expression of the c-myc gene in an EBV-immortalised B cell is sufficient to induce a tumorigenic phenotype, B cells from a common marmoset (Callithrix jacchus) were immortalised with EBV, transfected with a constitutively activated c-myc gene and inoculated into the host animals. Despite the cell line transfected with c-myc displaying enhanced growth characteristics, in vitro and in vivo experiments demonstrated that this was not sufficient to induce a tumorigenic phenotype. This supports our previous findings with EBV-immortalised human B cells transfected with an activated c-myc gene (Hotchin et al., 1990).

Inhibition of urethane leukaemia virus, a murine retrovirus, in mice by zidovudine.

The purpose of the study was to characterize in vivo an immunodepressive murine retroviral 'model' for the possible testing of drugs against HIV infection. Urethane leukaemia virus (ULV) injected into adult BALB/c mice (10(5) focus-forming units/mouse) caused a small, significant splenomegaly from 2 to at least 9 weeks after virus inoculation. Virus was also present in up to 60% nucleated splenocytes (XC 'infectious centre assay'). Effects on splenomegaly and virus in splenocytes were assayed following various regimens of zidovudine given as 0.5 mg/ml or 0.25 mg/ml in drinking water. Regimens included continuous treatment both before and after ULV, only before, and only after ULV inoculation. Zidovudine was also given for a limited period immediately after virus, or initiated after virus infection was established. Zidovudine given continuously at and following ULV infection completely prevented splenomegaly and virus expression in splenocytes. No other regimen was as effective; however, limited zidovudine treatment immediately after virus inoculation greatly reduced the effects of virus, while the same dose initiated after virus infection was established had only a small ameliorating effect. We conclude that ULV may prove to be a useful addition to other available murine systems, and this is discussed.

Establishment of immortalized primate epithelial cells with sub-genomic EBV DNA.

The genetic information in a sub-fragment of EBV DNA, designated p31 (containing less than a quarter of the viral genome and derived from a recombinant DNA cosmid library) allows epithelial cells from primary monkey and human kidney cultures to escape senescence under standard tissue culture conditions. A number of epithelial cell lines, designated M1/31, 483/31, 199/31 and HK/31, have been established and characterized following transfection of primary cells with p31 DNA. They share many properties, although morphologically they are not all identical. The cultures are immortalized but not fully transformed or tumorigenic. They appear to be phenotypically stable, although DNA hybridization studies indicate that genotypic alterations, including amplification, occur subsequent to transfection with p31 DNA and the establishment of a continuously proliferating epithelium. All cell lines consistently express high levels of cytokeratin 18 and varying amounts of cytokeratin 7, demonstrating their epithelial origin. From a single marmoset kidney (designated 199) a series of related immortalized cells, with subtle phenotypic differences, have been generated by p31 or sub-fragments of it. Although hallmarks of a "hit-and-run" mechanism are apparent in all of our studies, 2 different techniques (in situ hybridization or selection for cell survival in semi-solid media, followed by nucleic acid hybridization) show that, in late-passaged cultures, a small proportion of the cells still contain some viral DNA. The studies focus on genetic information within the BamHI A and I regions as being relevant to immortalization. The role of the EBV DNA fragment in the genesis of epithelial cell lines is considered.

Monoclonal antibodies from Epstein-Barr virus-transformed lymphocytes of common marmosets (Callithrix jacchus) immune to malaria.

The B lymphocytes of the common marmoset Callithrix jacchus can be immortalized by infection with Epstein-Barr virus (EBV) in vitro (Desgranges et al., 1976). C. jacchus is susceptible to infection with the blood stages of several species of malaria parasite including the line designated MVF1 (Mitchell et al., 1988) from which it recovers and shows immunity to reinfection. By exploiting these two phenomena, EBV-transformed, marmoset lymphoblastoid cell lines secreting antibodies to malaria parasite antigens have been generated and cloned. We believe this to be the first time that monoclonal antibodies (MAbs) have been raised from common marmosets. Since numerous and diverse human pathogens can infect this small primate in the laboratory, these methods may prove generally applicable for the generation of MAbs whose specificities derive from immune responses to infection.

Glomerulonephritis in common marmosets infected with Plasmodium brasilianum and Epstein-Barr virus.

Plasmodium brasilianum causes chronic quartan malaria in the common marmoset Callithrix jacchus, whereas Epstein-Barr virus (EBV) infection is followed by an infectious mononucleosis-like syndrome that resolves. We infected weanling marmosets with one or both of these pathogens. Timing of the infections influenced outcome. Six animals were simultaneously infected with both agents; four became seriously ill (with accompanying proteinuria and edema) and either died or were killed. Histopathology indicated that glomerulonephritis had developed. The two survivors had more-prolonged parasitemia than did animals infected with P. brasilianum alone, as did animals infected with EBV before P. brasilianum. Five of the six simultaneously infected animals had absent or low titers of antibody to Epstein-Barr viral capsid antigens when compared with the other EBV-infected animals. Our results suggest that combined infection may be part of the etiology of quartan malarial nephropathy.

Plasmodium vivax malaria in the common marmoset, Callithrix jacchus: adaptation and host response to infection.

Infection with Plasmodium vivax was established in splenectomized Callithrix jacchus marmosets by inoculation of parasitized blood from Aotus trivirgatus carrying the Vietnam Palo-Alto line of P. vivax. Subsequent blood passage through intact marmosets resulted in higher peak parasitaemias (about 1% of red cells infected) and the loss of stainable Schüffner's dots in infected cells. Primary infections with the adapted line were patent for 74 days or more, and induced both a substantial antibody response, as determined by indirect fluorescence, and some lymphocytosis, but no marked anaemia. Marmosets which had recovered from their primary infection (or in which it was drug-cured) suffered abbreviated patency with low-grade parasitaemia on re-infection.

Urinary cytology of a canine bladder carcinoma.

This is a study of a case of transitional cell carcinoma of the urinary bladder in a dog. Clinical and radiological signs were inconclusive. The morphology of cells exfoliated from the tumour was very similar to that of cells exfoliated from transitional cell carcinomas in human patients. On the basis of this information a diagnosis was made which was confirmed at post-mortem examination. The findings in this case report demonstrate the usefulness of this technique in the diagnosis of poorly differentiated transitional cell carcinoma.

Antiplatelet pyrimido-pyrimidines and metastasis.

The role of antiplatelet drugs in relation to their potential antimetastatic activities has been reviewed and the effects of two pyrimido-pyrimidine derivatives (RX-RA69 and RX-RA85) with strong antiplatelet activities investigated in metastasizing tumour models. The routes of administration and drug dosages were always chosen in such a way that good antiplatelet activities were obtained. RX-RA69 (20 mg/kg/day) given in the drinking water had no effect on spontaneous metastasis of Lewis lung carcinoma. RX-RA85 (20 mg/kg/day) did not influence spontaneous metastasis of B16 melanoma. On the other hand, giving RX-RA85 (8 mg/kg) daily i.p. to Lewis lung carcinoma bearing mice significantly increased the number of lung metastases but had no significant effect on primary tumour implant growth. Pretreating mice orally with 20 mg/kg RX-RA85 1 h before i.v. injection of B16 melanoma cells had no significant effect on lung colony number or distribution of extrapulmonary tumours while injecting the same dosage of RX-RA85 i.v. 1-2 h before tumour-cell injection decreased lung colony formation, but increased extrapulmonary tumour burden. This investigation like many others does not support the importance of platelets in metastasis formation.

Plasmodium brasilianum in the common marmoset Callithrix jacchus.

Chronic quartan malarial infection has been established in the common marmoset (Callithrix jacchus). Plasmodium brasilianum from a douroucouli monkey (Aotus trivirgatus) was used to infect splenectomized twin animals, passed to an intact animal, and then to 4 other intact adults, 2 pairs of twins. In 2 of the 4 latter animals there was continuing patency with parasitaemias of less than or equal to 0.5% parasitized erythrocytes for 30 weeks. The other 2 had lower initial levels of parasitaemia; in 1 of these parasitaemias remained low or subpatent. All marmosets developed lymphocytosis. One animal became ill 30 weeks after infection with anaemia, weight loss and mild proteinurea, the other 3 remained well. Histological examination showed minor changes in the kidneys; spleens of infected animals showed marked follicular hyperplasia and phagocytosis of pigment. The livers showed sinusoidal hypercellularity and pigment deposition and in splenectomized animals, a marked lymphoid follicular hyperplasia in the portal tracts.

Infectious mononucleosis-like response in common marmosets infected with Epstein-Barr virus.

Callithrix jacchus marmosets infected with Epstein-Barr virus (EBV) with or without concurrent treatment with cyclosporin A (CySA) remained healthy. Five marmosets given virus alone developed lymphocytosis and heterophile antibody. Antibody to EBV capsid antigens (VCA) appeared and remained at titers of 1:40-1:80 from 15 weeks onward. Two animals produced antibody to the R component of early antigens (EA) from six weeks onward. Five CySA-treated EBV-infected marmosets showed no increase in total lymphocyte counts; only two developed heterophile antibody. Four developed persistent antibody to the EA-R component. All developed antibody to VCA, and mean titers were higher than in animals given EBV alone. Antibody to VCA also appeared in animals given EBV into Waldeyer's ring. Because these responses to EBV resemble those of humans, C. jacchus may provide a useful model for exploring the potential of cofactors in inducing EBV-associated malignancy.

Cystoscopic examination of male and female dogs.

The use of cystoscopy is advocated as an aid to the early differential diagnosis of disease of the canine bladder. Techniques are described for carrying out urethroscopy and cystoscopy in both male and female dogs using modern medical diagnostic instruments. Males were examined with flexible paediatric bronchofibrescopes, which permitted urethroscopy and cystoscopy, but to obtain extensive biopsies or undertake cauterisation of the bladder surface with rigid endoscopes, a simple perineal urethrostomy was necessary. The bladder of females, on the other hand, was examined by cystoscopy and biopsied using standard rigid cystoscopes and resectoscopes.

Enhancement of the immune response to sheep erythrocytes in mice by phosphodiesterase-inhibiting dipyridamole derivatives.

Some potent phosphodiesterase (PDE)-inhibiting dipyridamole derivatives are able to increase the primary immune response in mice immunized with sheep red blood cells (SRBC). 10mg/kg/day of the most potent substance administered in the drinking water increased the number of plaque forming cells (PFC) in spleens of these mice by a factor of about 2 when the treatment was started after immunization. Pretreating the animals did not result in an enhancement of numbers of plaque forming cells. There was no increase in the background number of PFC.

Effects of lethal and non-lethal malaria on the mononuclear phagocyte system.

Estudou-se o efeito da infeccao causada por especie letal (Plasmodium berghei) e nao-letal (P. yoelii) de plasmodio sobre o sistema de fagocitos mononucleares de camundongo BALB/c. OP. yoelii causou maior e mais prolongada expansao e ativacao do sistema de macrofagos. As duas mais importantes populacoes de fagocitos esplenicosmacrofagos de polpa vermelha e da zona marginal - exibiam maior aumento do numero de celulas nesta infeccao. Durante a evolucao da malaria por P. berghei, o baco foi progressivamente ocupado por tecido hematopoietico e, na fase terminal da infeccao, observou-se significativa deplecao dos linfocitos e macrofagos esplenicos. Os dados apresentados indicam que a evolucao da malaria depende do tipo de interacao entre o plasmodio e o sistema de fagocitos mononucleares