Large-scale purification of Epstein-Barr virus membrane antigen gp340 with a monoclonal antibody immunoabsorbent.

A purification method has been elaborated to isolate Epstein-Barr (EB) virus membrane antigen, gp340, in milligram amounts. The gp340 was prepared from detergent extracts of B95-8 cells by affinity chromatography with a monoclonal antibody immunoabsorbent. Bound material was eluted and the eluate, consisting of 50% gp340, was then fractionated by gel filtration. The final gp340 product was antigenically active and 95% pure. The purification method was found to be rapid and reproducible with no loss of the ability of the immunoabsorbent to retain gp340 after repeated elution. The procedure provides suitable material to permit the detailed structural analysis of gp340 necessary for both vaccine design and for the investigation of the role of gp340 in immunity to EB virus infection.

Monoclonal antibody 2C5: a marker for a subpopulation of small neurones in rat dorsal root ganglia.

Monoclonal antibody 2C5 labels a subset of dorsal root ganglion neurones in the rat. The cell sizes of these neurones fall within the range for the small dark cell population and the antibody labels between a half and two-thirds of the neurones in this size range. A subpopulation of small neurones was also labelled in the trigeminal and vagal ganglia. Other sites of immunoreactivity in the central nervous system are the region of the substantia gelatinosa of the spinal cord, fibres in Lissauer's tract, the tractus solitarius and the tuberculum olfactorium. These sites are consistent with the antigen being expressed by the central processes of primary afferent neurones. It is suggested that the size distributions of 2C5-positive dorsal root ganglion neurones and the pattern of 2C5 immunoreactivity within the spinal cord indicate that the labelled cells may be neurones with peripheral C fibers. Outside the nervous system the antigen is expressed in a number of specific cell types within a variety of organs. These include some pancreatic acinar cells, parietal cells of the gastric mucosa, some cells in taste buds, Leydig cells of the testis, scattered cells in lymph nodes and lung alveoli, some renal tubules, the epithelial lining of the fallopian tube, the epithelium covering the ovary and certain cells in the basal layer of the epidermis.

Lineage and non-lineage related expression of an anti-teratocarcinoma monoclonal antibody reactivity in the post-implantation embryo and adult mouse.

Distribution of monoclonal antibody 2C5 reactivity is studied in post-implantation embryonic material and the adult mouse. Reactivity of 2C5 monoclonal antibody, raised against PC13 embryonal carcinoma, is reduced on in vitro differentiation of teratocarcinoma stem cells. 2C5 antibody reacts with embryonic and extraembryonic endoderm in the post-implantation egg cylinder. Rathke's pouch at the tenth day and primordial germ cells at the thirteenth day of gestation express 2C5 antigen. In the adult, detectable 2C5 antigen expression is limited to the thymus, pituitary body and female reproductive tract. The apparent non-lineage related expression of 2C5 monoclonal antibody and the reactivities of other monoclonal antibodies are discussed.

A highly sensitive enzyme-linked immunosorbent assay to quantitate antibodies to Epstein-Barr virus membrane antigen gp340.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of antibodies to Epstein-Barr (EB) virus membrane antigen (MA) glycoprotein, gp340, in tamarins. The assay was found to be a thousand-fold more sensitive than conventional indirect immunofluorescence tests and consequently it was possible to follow accurately the sequential production of specific antibodies to gp340 by tamarins during a course of immunization.

Leukocyte origin and profile in follicular aspirates at oocyte retrieval.

BACKGROUND: Follicular aspirates represent a snapshot in time of conditions within the follicle at oocyte retrieval in women undergoing in vitro fertilization and embryo transfer. This clinical material has been much investigated and yet its cellular composition remains unclear. In this study we investigated the origin and profile of leukocytes found within follicular aspirates. METHODS: We performed morphological and immunohistochemical analyses of follicular aspirates and peripheral blood obtained concurrently at oocyte retrieval. RESULTS: There was no correlation between erythrocyte and leukocyte numbers in follicular aspirates. The profile of leukocyte subtypes within follicular aspirates was variable and differed significantly from the peripheral circulation in a significant proportion of the analysed samples. A subset of follicular aspirates displayed a marked increase in monocytes/macrophages and an apparent concomitant reduction in polymorphonuclear leukocytes compared with peripheral blood. CONCLUSIONS: Leukocytes within follicular aspirates cannot be accounted for solely as a result of blood vessel damage during oocyte retrieval. The variation in leukocyte subtypes observed in some follicular aspirates may reflect a coordinated infiltration of these cells, characteristic of progressive inflammatory responses in other systems. The possibility that leukocyte variation is indicative of follicular maturation deserves further investigation due to its potential relevance in optimizing oocyte selection.

Cosegregation of monoclonal antibody reactivity and cell behaviour in the mouse preimplantation embryo.

Expression of an antigen, recognized by a monoclonal antibody raised against PC13 embryonal carcinoma, is described in mouse preimplantation embryogenesis. The antigen is found in the cytoplasm of ovulated ova and is first noted on the cell surface of the 1-cell embryo 20 h post-ovulation. Surface labelling of blastomeres is uniform until the 8-cell stage when antigen expression becomes polarized along the radial axis of the embryo. Two major populations of blastomeres are distinguishable on division to the 16-cell morula. Dissociation of morulae in calcium-free medium yields large, polar, antigen-positive cells and small apolar cells with reduced levels of detectable antigen. A third, minor population of small, antigen-negative cells is also found in vivo. Large and small blastomeres differ in their ability to relocate within the embryo when aggregated with intact 16-cell-stage embryos. The small blastomeres of the 16-cell morula contribute significantly to the inner cell mass while the large antigen-positive cells are found only in the trophectoderm.

Interferon synthesis in the early post-implantation mouse embryo.

A qualitative bioassay was adapted and used to determine the ability of the early post-implantation mouse embryo to synthesise interferon. Interferon production was not seen in any embryo tissue in the absence of an inducer and could only be detected in virus-induced tissue from the early 7th day of development. This induced interferon synthesis was initially confined to the trophoblast of the early 7th day embryo. It was then found in tissues of both trophoblast and inner cell mass origin in the early 8th day, and subsequently, in derivatives of the embryonic ectoderm in the 13th-day embryo.

Protection of cottontop tamarins against Epstein-Barr virus-induced malignant lymphoma by a prototype subunit vaccine.

Epstein-Barr (EB) virus is one of the five herpesviruses of man. Strong links between this agent and the chain of events causing two human cancers, endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, have long been evident (reviewed in ref. 1). Because of this, and because of the very high incidence of nasopharyngeal carcinoma in certain large populations, it was suggested in 1976 that a vaccine should be developed against EB virus to prevent infection and thereby reduce tumour incidence amongst those at risk. The virus-determined membrane antigen (MA) was proposed as immunogen because it was known to elicit naturally occurring virus-neutralizing antibodies in man and because analogous antigens had been shown to act as effective experimental vaccines for preventing the herpesvirus-induced lymphomas of Marek's disease in chickens. Progress has been achieved in defining, quantifying and preparing MA molecules, and in enhancing their immunogenicity; a sensitive assay for antibodies to MA has been elaborated. Here we report that isolated cell membranes expressing MA, or purified MA glycoprotein of relative molecular mass (Mr) 340,000 (gp340), have been used to vaccinate cottontop tamarins (Saguinus oedipus oedipus), and that animals receiving either preparation were protected against the effects of a 100% tumour-inducing challenge dose of EB virus.

Not all potently neutralizing, vaccine-induced antibodies to Epstein-Barr virus ensure protection of susceptible experimental animals.

Cottontop tamarins have been immunized with a high molecular weight, Epstein-Barr (EB) virus membrane antigen (MA) glycoprotein (gp340) separated by monoclonal antibody immunoaffinity chromatography (MCABgp340). Specific antibody production was monitored by immunofluorescence, a highly sensitive enzyme-linked immunosorbent assay (ELISA), and virus neutralization tests, and was found to reach high titre after 4/5 inoculations. The animals were challenged with a 100% lymphomagenic dose of EB virus but despite possessing powerful neutralizing antibodies were not protected against tumour causation by the virus. This result contrasts with that of earlier experiments in which tamarins with neutralizing antibodies induced by gp340 prepared by a molecular weight-based method (MWgp340) were protected. The reasons for this difference in protection associated with vaccine molecules prepared in different ways are discussed together with the need for parameters other than neutralizing antibody for use in the assessment of subunit immunogens against EB virus.

Damage to surfactant-specific protein in acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) develops in association with many serious medical disorders. Mortality is at least 40%, and there is no specific therapy. A massive influx of activated neutrophils, which damage pulmonary vascular endothelium and alveolar epithelium, leads to alveolar oedema and pulmonary surfactant dysfunction. In-vitro studies show that neutrophil elastase can cleave surfactant-specific proteins and impair surfactant function. If this happens in vivo in ARDS, the response to surfactant therapy will be limited. METHODS: Samples of pulmonary surfactant were obtained from the lungs of 18 patients with ARDS and six healthy controls by bronchoalveolar lavage. We separated proteins in these samples according to molecular weight by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). We then used western blotting with monoclonal antibody E8 to detect the major surfactant-specific protein A (SP-A). FINDINGS: By contrast with controls, 14 of 18 patients had evidence of in-vivo damage to SP-A that resembled damage caused to SP-A when it is cleaved by neutrophil elastase. Controls showed a single band of normal dimers at 66 kDa, whereas 14 of 18 patients showed multiple bands at 66 kDa, 55 kDA, and 30-36 kDa, and six showed additional bands at 36-40 kDa. INTERPRETATION: Direct damage to surfactant-specific proteins occurs in lungs of patients with ARDS, probably by proteolysis. Trials of protein-containing therapeutic surfactant are in progress in ARDS, and our results indicate that the frequent failure to maintain response may result from continuing damage to surfactant by products of activated neutrophils. A combination of surfactant and antiprotease therapy may improve therapeutic prospects.