Photodynamic therapy targeted to tumor-induced angiogenic vessels.

Cancer photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) may be effective not only by being directly cytotoxic to tumor cells, but also by being cytotoxic to the endothelium of tumor neovasculature. In the present study, we investigated the effect of PDT with an experimental liposomal formulation of BPD-MA on tumor-induced angiogenic vessels using a murine dorsal air sac model. First, hemostasis of neovasculature was examined by varying the regimen of PDT. Laser irradiation at 15 min after injection of 2 mg/kg liposomal BPD-MA (15 min PDT) caused complete blocking of blood flow in neovasculature. In contrast, PDT did not inhibit blood flow when the irradiation occurred 3 h after the injection of liposomal BPD-MA (3 h PDT). Next, the antitumor effect of PDT on Meth A sarcoma-bearing mice was investigated by using the hemostasis-inducing regimen. Tumor growth was strongly inhibited after the 15 min PDT with BPD-MA at a dose of 0.5-2 mg/kg. In contrast, 3 h PDT with BPD-MA at a dose of 2 mg/kg suppressed tumor growth only partially. The current study indicates that 15 min PDT causes strong suppression of tumor growth, perhaps through damaging endothelial cells in the tumor neovasculature rather than through a direct cytotoxic effect on tumor cells.

Comparative immunogenicity studies on Epstein-Barr virus membrane antigen (MA) gp340 with novel adjuvants in mice, rabbits, and cotton-top tamarins.

The effectiveness of immunisation of mice, rabbits, and cotton-top tamarins with small amounts of EB virus MA glycoprotein gp340 , incorporated into artificial liposomes, has been compared using various routes of injection with or without additional adjuvants. Liposomes containing gp340 gave specific high titre antibodies after i.p. or i.v. administration, and the addition of lipid A to the liposomes resulted in a significant enhancement of the response. Antibodies generated by the above procedure were virus neutralising and bound gp340 specifically. These findings indicate an advantageous approach for use with a prototype vaccine for the prevention of EB virus infection.

Purification and properties of the gp340 component of Epstein-Barr virus membrane antigen in an immunogenic form.

Preparative SDS-polyacrylamide gel electrophoresis has been used in the purification of the gp340 component of Epstein-Barr (EB) virus-determined membrane antigen (MA), in tractable quantities, from the B95-8 marmoset lymphoblastoid cell line. Successful renaturation of the purified molecule was achieved. This procedure gave a 50-fold increase in the recovery of antigen compared to conventional techniques. The data suggest that the antigenic sites recognized by human sera containing antibodies to MA are largely confined to the protein portion of the molecule. An eightfold improvement in the yield of gp340 was obtained when B95-8 cells were cultured in the presence of 12-O-tetradecanoyl-phorbol-13-acetate. Gel filtration studies indicate that the major polypeptide components of MA are not associated in detergent solution. Immunization of rabbits with purified and renatured gp340 resulted in the generation of high-titre antisera which were specific for gp340, demonstrating that antigen prepared by this procedure is suitable for further evaluation as an experimental vaccine against EB virus infection.

Purified Epstein-Barr virus Mr 340,000 glycoprotein induces potent virus-neutralizing antibodies when incorporated in liposomes.

The purified Mr 340,000 glycoprotein component of Epstein-Barr (EB) virus-induced membrane antigen complex incorporated into liposomes was shown to be a potent immunogen in mice. High-titer antisera were induced that (i) are specific for membrane antigen components without absorption, (ii) bind the antigens induced by three different EB virus isolates, and (iii) neutralize the ability of the virus to transform fetal cord blood lymphocytes in vitro. The development of this immunogenic form of purified antigen provides an important step towards a potential subunit vaccine against Epstein-Barr virus infection.

Quantification of an Epstein-Barr virus-associated membrane antigen component.

A method is described for the preparation of a 125I-labelled membrane antigen (MA) component (gp340) from B95-8 cell membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Good yields of antigenic material were obtained when renaturation of the [125I]gp340 was carried out by removal of SDS in the presence of urea and subsequent removal of the urea. The availability of purified, radiolabelled gp340 has provided the essential basis for the development of a radioimmunoassay which, for the first time, permits quantification of this antigen. The assay has been used to demonstrate that cell membrane MA is a better source of gp340 for large-scale work than is the Epstein-Barr virus envelope and to measure the increase in expression of gp340 following treatment of cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA).

Antigenic differences between the membrane antigen polypeptides determined by different EB virus isolates.

The antigenic specificities of the membrane antigen (MA) complex of three Epstein-Barr (EB) virus-producing cell lines have been compared by complete absorption of an anti-MA antiserum with P3HR-1 cells, followed by testing for residual anti-MA antibody activity against B95-8 and QIMR-WIL cells. Indirect membrane immunofluorescence showed that the absorbed serum, which had lost the capacity to bind P3HR-1 cells, nevertheless gave bright staining on a small proportion of B95-8 cells. SDS-polyacrylamide gel electrophoretic analysis of 125I-labelled MA polypeptides showed that the absorbed serum was able to isolate MA polypeptides from both B95-7 and QIMR-WIL cells, but not from P3HR-2 cells. The results demonstrate that there is substantial sharing of antigenic specificities between the MA determined by three isolates of EB virus, and some antigenic divergence.

Observations on the EB virus envelope and virus-determined membrane antigen (MA) polypeptides.

Experiments have been carried out to identify the polypeptide components of the Epstein-Barr (EB) virus-determined membrane antigen (MA) complex. Cells were radioiodinated using lactoperoxidase and the 125I-labelled surface antigens, released by Triton X100, were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) after complexing either with human sera having anti-MA activity or with a rabbit antiserum to EB virus. Using cells carrying EB virus isolated from four different conditions, including for the first time nasopharyngeal carcinoma (NPC), we have demonstrated four major polypeptides. One, with a molecular weight of 85,000, was remarkedly constant irrespective of the cell line from which it came; two, with molecular weights varying from 240,000 to 270,000 and from 320,000 to 340,000, showed minor differences in mobility apparently depending on the species of origin of the cells (human or marmoset), rather than disparity between strains of virus. A fourth component, of 160,000 daltons, was found on only two of the cell lines studied. In addition, it has been shown for the first time that the same polypeptides composing the MA complex are present on the viral envelope itself. The fact that the rabbit antiserum to EV virus recognized only the two highest molecular weight MA components, yet showed virus-neutralizing activity, indicates the importance of these two polypeptides for use in a vaccine designed to induce virus-neutralizing antibodies.

Differentiation antigens on normal and Abelson virus transformed lymphocytes.

Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.

IgG anti-hapten antibody secretion in vitro commences after extensive precursor proliferation.

We have investigated whether the intermediate cells that arise in primed mice after boosting require further cycles of division in culture before maturation into IgG antibody secreting cells. Killing of dividing cells between days 1--4 in culture, by exposure to BUdR-uv irradiation ablated the high IgG response observed on day 5 in control cultures. After T cell removal and replacement by a soluble factor (TRF) similar results were obtained. Thus B cell division over an extended period occurs prior to the appearance of IgG secreting cells. Furthermore, autoradiography of plaques from cultures briefly exposed to [3H]thymidine before harvest showed that some antibody secreting cells were synthesizing DNA at the time of assay.

Non-specific factor replaces T cells in an IgG response to soluble antigens.

The antigen and T-cell requirements for the final stages of proliferation and maturation of DNP-KLH primed and boosted mouse spleen cells into IgG antibody secreting cells have been studied in vitro. The requirement for free antigen ceases after 24-48 h in vitro. The carrier-specific T-cell requirement for triggering of activated B cells by a soluble antigen (DNP-KLH) can be replaced in T cell-depleted cultures by non-antigen specific T cell-replacing factors (TRF). However, if the carrier protein is changed, TRF restores the IgG response of T cell-depleted cultures only if antigen is presented to B cells in particulate form, e.g. on the surface of macrophages, or in the presence of small amounts of antibody against the carrier protein. Thus, direct interaction between soluble protein and B cells is not sufficient to allow TRF to effectively replace specific T cells. Since TRF must be added at the start of culture, the initiation of B-cell maturation into IgG secretion by TRF occurs during B-cell proliferation, and is followed by further proliferation before IgG antibody can be detected.

The immunoglobulin class of anti-hapten antibody secreted during secondary responses in vitro and in vivo.

A comparison has been made of the in vitro and in vivo response of primed mouse spleen cells to the hapten DNP. The responses were analysed in terms of six classes (sub-classes) of humoral antibody directed against the cross-reacting hapten TNP. By comparison with the response in intact mice the adoptive secondary response is delayed by 3 days in addition to being somewhat lesser in magnitude. The timing of the response in vitro is similar to that observed in intact mice. The preponderant class in all three responses was gammaG1 with gammaA and gammaG3 secreting cells consistently comprising the smallest proportion of the total of antibody-secreting cells.

B-memory cells can be stimulated by antigen in vitro to become IgG antibody-secreting cells.

Whereas DNP-KLH primed mouse spleen cells fail to show the high IgG anti DNP levels characteristic of an anamnestic immune response when cultured for 5 days under Mishell-Dutton conditions, we show here that such a response can be observed after 8--10 days in vitro using modified Marbrook culture vessels. The kinetics of this secondary response in vitro resemble that described elsewhere for the adotpive secondary immune response in irradiated recipients, and show that insignificant numbers of IgG PFC can be expected before 6 days of culture. This timing is governed by the maturation state of B cells at the start of culture, and was not accelerated by the addition of recently boosted carrier primed spleen cells. We conclude that the deficit in IgG PFC numbers that characterizes Mishell-Dutton cultures of cells primed 2--4 months previously is due to the shortage of time during which such cultures can maintain lymphocyte division and maturation.

IgG response in vitro. I. The requirement for an intermediate responsive cell type.

We have studied a secondary IgG anti-dinitrophenyl response in dissociated mouse spleen cell cultures which is comparable in magnitude to the responses detected after adoptive transfer. The data indicate that the responsive precursor cell is not a recirculating memory cell but is an intermediate B cell type which appears in vivo after antigen challenge of primed mice. Both antigen and T helper cells are required for in vitro stimulation of the intermediate cell type into the division and maturation steps leading to IgG antibody secretion. The buoyant density of precursor cells has been analyzed by bovine serum albumin density gradient centrifugation, and is consistent with their recent origin by division from memory cells.