Biomedical subjects
W Z Wei
Publications and source records attributed to W Z Wei.
PEPMOTIF: a program for locating class I major histocompatibility complex restricted peptides in protein sequences.
PEPMOTIF is a computer program which analyzes protein sequences for the occurrence of peptides up to ten residues in length which contain motifs presented by particular class I major histocompatibility complexes. Any peptide motifs defined by the user can be identified in a protein sequence of interest. PEPMOTIF generates a listing of all motif-containing peptides found in the protein, and two modes of data output are provided: (1) direct printout, or (2) storage in a text file on disk.
Systematic identification of H-2 Kd binding peptides and induction of peptide specific CTL.
Most peptides with putative MHC I restricted sequence motifs do not bind to the corresponding MHC I nor induce cytolytic T cells. There exist additional constraints which limit peptide binding and immunogenicity. To identify immunogenic peptides in novel protein sequences, it will be necessary to first evaluate peptide binding to MHC I. In this study, a soluble single chain fusion protein SC-Kd was used to evaluate potential Kd binding peptides from the sequences of mouse mammary tumor virus gag and env proteins. A total of 27 peptides were identified which displayed the reported Kd restricted motif. Of the 27 peptides, six demonstrated strong to moderate binding to SC-Kd. The strongest binding peptides expressed tyrosine or phenylalanine at position 2 and leucine at the C-terminus. The capability of MMTV peptides to induce CTL corresponds to their SC-Kd binding activity. Of the six peptides that demonstrated moderate to strong binding, five induced CTL in BALB/c mice. These peptides induced CTL after 1-3 in vivo immunizations followed by 5 day in vitro stimulation. Furthermore, a single in vitro stimulation of naive lymphocytes with strong-binding G425 was sufficient to induce significant CTL activity. Weak or non-binding peptides did not induce CTL. Therefore, peptide binding to SC-Kd is a predictive indicator of CTL inducing activity.
Activation of natural killer cells by mouse mammary tumor virus C4 in BALB/c and T-cell receptor V beta 2-transgenic mice.
Lymphocytic infiltrates of BALB/c C4 hyperplastic alveolar nodule (HAN) have elevated natural killer (NK) activity, which correlates positively with the progression of C4 HAN to tumor: C4 HAN produces an infectious mouse mammary tumor virus, MMTV(C4), which encodes a superantigen that activates and deletes T-cells with the V beta 2 segment in the T-cell receptor. In this report, NK activation by both MMTV(C4) and MMTV(C4) superantigen was tested. NK activity was measured in naive BALB/c mice, BALB/c mice depleted of V beta 2+ T-cell, or V beta 2-transgenic mice after they received injections of either purified MMTV(C4) or MMTV(C4)-infected splenocytes. Elevated NK activity was observed in BALB/c mice receiving MMTV(C4) or MMTV(C4)-infected splenocytes. Depletion of V beta 2+, but not V beta 8+, T-cells by specific anti-V beta hybridoma before injection of MMTV(C4)-infected cells reduced but did not eliminate NK activation. NK activation in V beta 2-transgenic mice occurred before massive CD4 T-cell deletion took place and was more pronounced than that in the nontransgenic littermates. These results indicate that MMTV activates NK cells through superantigen-dependent and -independent pathways and supports the role of MMTV(C4) in the augmented NK activity observed in C4 HAN infiltrates. The progression of C4 HAN to tumor represents a model system for the analysis of how tumorigenesis may be affected by lesion-associated viruses.
Deletion of CD4+ T cells and thymocytes by apoptosis in mouse mammary tumor virus (C4)-infected V beta 2 transgenic mice.
Mouse mammary tumor virus MMTV (C4) encodes a V beta 2-specific superantigen. In V beta 2 transgenic (TG2) mice more than 98% of peripheral T cells express V beta 2. Infection of Tg2 mice with MMTV (C4) at birth through their mothers' milk or at 6-8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4+ T cells and suppressed thymopoiesis. The number of peripheral CD8+ T cells was not affected in neonatally infected mice. In older mice injected with MMTV (C4), splenic CD8+ T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV (C4). Thymocytes which expressed high level CD3 or V beta 2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31% of CD4+ T cells from MMTV (C4)-infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow-cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV (C4)-infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4+ T cell and thymocyte deletion by MMTV (C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen-induced apoptosis.
A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules.
We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.
[Simultaneous determination of vitamin B1 and vitamin C in aqueous solution with piezoelectric crystal quartz sensors].
According to the response properties of piezoelectric crystal quartz sensors to the solution conductivity the frequency shift response was derived to show a linear dependence on the concentrations of vitamin B1 (VB1) and vitamin C (VC). This was experimentally verified and a calibration model for simultaneous determinations of the two vitamins was established. The usefulness of the technique was evaluated by quantitation of mixtures of unknown composition using common multiple linear regression (MLR) and partial least squares (PLS). The average relative standard deviations for six samples were 4.46% for VB1 and 6.63% for VC with MLR; and 1.97% for VB1 and 2.74% for VC with PLS, respectively.
Mouse mammary tumor virus associated antigens and superantigens--immuno-molecular correlates of neoplastic progression.
There is a large body of literature on immune reactivity to mouse mammary tumor virus (MMTV) during mammary tumor progression. It is a general consensus that MMTV is antigenic and elicits cell-mediated and humoral immune responses. In addition, activation of non-specific inflammatory cells has been observed in various mouse preneoplastic and neoplastic mammary lesions. How these non-conventionally activated inflammatory cells affect tumor progression is a subject of debate. The discovery of MMTV associated superantigens solved certain long standing mysteries and may provide new insight into the cause of these unusual host responses. In this article, we will discuss how superantigen associated secondary immune responses may contribute to mammary tumor progression and the possible impact superantigen may have on specific anti-MMTV reactivity.
Adoptive transfer of V beta 2-deleting activity with host cells from mice implanted with C4 preneoplastic hyperplastic alveolar nodules.
Selective deletion of mature peripheral V beta 2+ T-cells was observed in BALB/c mice implanted with the syngeneic C4 preneoplastic hyperplastic alveolar nodule (HAN) but not in mice with sham surgery (W. Z. Wei, R. Ficsor-Jacobs, S. J. Tsai, and R. Pauley, Cancer Res., 51:3331-3333, 1991). We now report the participation of host cells in that process. Peripheral V beta 2+ T-cells were reduced by 50% or more 4 weeks after an i.v. injection of 10 x 10(6) spleen cells from C4 HAN-bearing mice. Both T- and B-cell-enriched splenocytes mediated V beta 2 deletion. Secondary adoptive transfer of splenocytes from the recipients of the primary adoptive transfer also resulted in V beta 2+ T-cell deletion. The splenocytes lose V beta 2-deleting activity after 500 rad irradiation. V beta 2 deletion induced by either C4 HAN or splenocytes was more profound in CD4+ than in CD8+ T-cells. Loss of V beta 2+CD4+ T-cells was observed 5 days after the adoptive transfer of splenocytes, whereas V beta 2+ CD8+ cells were not reduced until day 9 or later. The differential rate of V beta 2+ CD4+ and CD8+ T-cell reduction continued for at least 7 weeks after the adoptive transfer. The pattern of V beta 2 deletion and the sequence of T-cell loss is similar in the recipients of C4 HAN or of adoptively transferred splenocytes. Southern blot analysis demonstrates non-germ line Mtv or MMTV proviral DNA in C4 HAN. Splenocytes of C4 HAN-bearing mice express a higher level of 1.7-kilobase long terminal repeat transcript than normal BALB/c splenocytes, suggesting a role for a unique Mtv/MMTV provirus in V beta 2 deletion.
Elimination of V beta 2 bearing T-cells in BALB/c mice implanted with syngeneic preneoplastic and neoplastic mammary lesions.
The effect of growth of BALB/c C4 preneoplastic and neoplastic mammary lesions on the host T-cell repertoire was investigated. T-cells with specific V beta regions (V beta 2, -6, -8.1-2, -11, and -14) in the T-cell receptors were enumerated in C4 hyperplastic alveolar nodule (HAN)-infiltrating lymphocytes and lymph node cells from both C4 HAN and C4 tumor-bearing mice by flow cytometry with V beta-specific monoclonal antibodies. Growth of C4 HAN and tumor induced selective deletion of peripheral V beta 2+ T-cells. Elimination of V beta 2+ T-cells [7.2 +/- 0.8% (SD) of normal lymph node T-cells] by C4 HAN was biphasic and irreversible. Approximately one-half of the V beta 2+ T-cells were lost within the first month of C4 HAN implantation. Further reduction of V beta 2+ T-cells took place after another 3 months, at which time the level of V beta 2 was reduced to 1.2 +/- 0.1% of the total T-cell population. V beta 2 deletion occurred in BALB/c mice which had been implanted with C4 HAN at either 3 weeks or 2 months of age. Loss of V beta 2+ T-cells was not reversed by removal of C4 HAN. C4 tumor also induced V beta 2+ T-cell deletion. These results demonstrate a novel V beta 2 deleting activity expressed by C4 mammary lesions and suggest that during mouse mammary tumorigenesis, a unique "superantigen" is expressed which can cause profound systemic changes in the T-cell repertoire.
Two-dimensional gel analysis of polypeptides from normal, preneoplastic and neoplastic mouse mammary tissues.
High-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) was employed to reveal tumor-associated polypeptide changes, using the BALB/c C4 line mouse mammary model system, for which phenotypic and immunogenic alterations accompanying tumor progression are well defined. In the first set of experiments, polypeptide patterns from 20 micrograms whole tissue lysates of normal mammary gland, C4 preneoplastic hyperplastic alveolar nodule outgrowth (HAN) and spontaneous tumor from C4 HAN were compared. In order to normalize for differential cellularity and extracellular protein content in the whole tissues, our analysis included polypeptide patterns from serum, increased concentration of protein from whole normal mammary gland, and primary cultures of epithelial cells from normal gland, HAN and tumor. Using a computer-based image-analysis system, 90 polypeptides were identified in C4 tumor that were absent in C4 HAN, normal mammary gland and serum. None of the 90 polypeptides could be shown to represent a definite qualitative change in the protein composition of tumor epithelium as they were found to be either present in a higher concentration of protein from whole normal gland, or present in the primary epithelial culture from HAN, or absent in the primary epithelial culture from tumor. Conversely in the second set of experiments, when epithelial cultures were used as the starting point for comparisons to locate tumor-associated polypeptides, none of the 15 polypeptides that were present in cultures from three different tumors, and absent in the culture from normal mammary gland was specific to C4 tumor, as they were present in whole tissues of normal gland. Thus our experimental approach detected significant quantitative but no qualitative polypeptide changes in whole tumor tissue, or in tumor-derived epithelial cell cultures. This finding may reflect the limitations of the two-dimensional PAGE method, and warrants caution in the use of such gel analysis alone to identify tumor-associated proteins.
Improved anti-tumor reactivity with monoclonal anti-idiotypic antibody conjugated to syngeneic mouse red blood cells.
An anti-idiotypic (anti-Id) antibody (Ab), by resembling an epitope on an antigenic molecule may serve as a surrogate antigen and induce specific immune reactivity to the natural antigen. Anti-Id Ab are often conjugated to a foreign protein, such as keyhole limpet hemocyanin (KLH) to elicit a stronger response. However, KLH, a potent antigen itself, may provoke unnecessary and undesirable immune reactivity. In this study, syngeneic mouse red blood cells (RBC) were tested as carriers for anti-Id immunization. In a preliminary experiment, BALB/c mice were immunized with normal rat immunoglobulin G (NRIg)-MRBC or NRIg-KLH. Anti-NRIg responses induced by 2-0.2% of NRIg-MRBC (4-0.4 micrograms of NRIg) superseded those induced by NRIg-KLH (50 micrograms of NRIg). Immunization with NRIg-MRBC did not result in autoreactivity against self antigens on autologous RBC. Monoclonal antibody 2F10, which resembles an epitope on mouse mammary tumor virus (MMTV) envelop protein Gp52, was conjugated to MRBC and used as a surrogate antigen for Gp52. Immune sera were tested in cytotoxicity assay against MMT 68H which expresses Gp52. Between 2-4 weeks after the immunization, specific cytotoxic antibodies were detected in mice immunized with 2.0%, 0.2% and 0.02% 2F10-MRBC, with 0.2% 2F10-MRBC being the most effective dose. 2F10 immune sera did not react with autologous RBC. Therefore, MRBC are at least as effective, and may be more effective than KLH as carriers for Ig immunization. Repeated treatment will be feasible since MRBC are not immunogenic.
Correlation of natural killer activity with tumorigenesis of a preneoplastic mouse mammary lesion.
Tissue infiltrating lymphocytes isolated from the preneoplastic mouse mammary hyperneoplastic alveolar nodule (HAN) tissue line C4 express high levels of natural killer (NK) activity, which gradually wanes as spontaneous tumors develop (W. Z. Wei and G. Heppner. Br. J. Cancer, 55: 589-594, 1987). Experiments were performed to test whether modulation of NK cell activity would be associated with altered progression of HAN to tumor. Administration of polyinosinic-polycytidylic acid, which activates NK activity but does not directly affect mammary epithelial cell growth, to HAN-bearing mice enhanced tumor progression, as measured by a decrease in the latency period and increase in the incidence of mammary adenocarcinomas developing in the HAN implants. Antiasialo GM1, which reduces NK activity, reduced tumor progression. The net effect of indomethacin, which may inhibit mammary epithelial cell growth but enhances NK cell function, was to prolong the latency period of tumor development. However, this effect was reversed by interleukin 2, which activates NK cells. These findings suggest that NK activity may provide positive signals for progression of preneoplastic mammary lesions to frank neoplasia.
Modulation of lymphocytic infiltrates in C4 mouse mammary tumors.
Explore the source record for details and available documents.
Expression of epithelial-like markers and class I major histocompatibility antigens by a murine carcinoma growing in the mammary gland and in metastases: orthotopic site effects.
A spontaneous murine mammary carcinoma, designated SP1, grew more aggressively in the mammary gland than in the subcutis exhibiting a 10-fold lower 50% lethal tumor dose and the ability to metastasize spontaneously from the orthotopic mammary gland site. The appearance of metastasis could be abrogated by resection of the primary tumor up to 21 days postinjection, arguing against the possibility that metastasis occurred due to trauma of the injection and/or healing processes. In addition, tumor cells recovered from lung metastases exhibited an increased ability to metastasize when reinjected into either the s.c. or mammary sites. Tumor cells from lung metastases showed low levels of Class I major histocompatibility (MHC) antigens, like the parental SP1 cells, but were found to express differentiation markers typical of normal basal and luminal mammary epithelium. SP1 tumors expressed increased Class I MHC antigens, as well as high levels of basal and luminal breast epithelial markers, within 7 days of implantation into the mammary gland. On the other hand, SP1 tumors growing in the subcutis never expressed increased Class I MHC levels and expressed the epithelial marker antigens at lower levels and not until at least 21 days of growth. Removal of host epithelium by cauterization of the mammary bud at 3 weeks had no effect on the increased growth, metastasis and acquired heterogeneity of MHC and epithelial associated antigens, suggesting that the mammary gland stroma was responsible for the observed phenomenon. These findings suggest that the mammary gland either selects distinct tumor subpopulations, or induces a phenotypic change leading to tumor progression and the generation of metastatic subpopulations.
Tumor infiltrating lymphocytes of spontaneous versus transplanted mouse mammary tumors.
Tumor infiltrating lymphocytes (TIL) were isolated by centrifugal elutriation from C4 mouse mammary tumors and characterized with regard to phenotype and natural killer (NK) activity. Tumors that had arisen spontaneously in preneoplastic hyperplastic alveolar nodules and tumors that had been passaged one to two times in either naive or presensitized mice were studied. Mice were sensitized by limited s.c. tumor growth and subsequent surgical removal of the tumor. The total numbers of T or B cells in the infiltrates were similar in spontaneous tumors and in passaged tumors from either naive or sensitized mice. The ratio of L3T4-positive to lyt-2-positive cells was reduced, however, from 1.10 +/- 0.2 in spontaneous tumors to 0.53 +/- 0.28 or 0.48 +/- 0.04 in passaged tumors from untreated or sensitized mice. The site of tumor implantation, whether intramammary fat pad or s.c., did not affect the profiles of the infiltrates. The TIL from both spontaneous and passaged tumors demonstrated enhanced NK activity relative to peripheral lymphoid cells. The TIL of passaged tumors sensitized mice, however, had lower NK activity than those from naive mice.
A new assay to measure monoclonal antibody-dependent complement or macrophage-mediated tumor growth inhibition.
A new in vitro assay has been developed to measure the inhibition of tumor growth by antibody and complement or by antibody and macrophages. Tumor cells (1 X 10(5) cells) or a mixture of tumor cells (1.5 X 10(4) cells) and macrophages (1.5 X 10(5) cells) are immobilized in a 1 microliter agarose droplet. Antibody is added to the medium bathing the agarose droplet. Complement is also added to wells containing droplets with tumor cells alone. The area covered by tumor cell monolayer is measured non-destructively from day 0 to day 7 with a split image tracing device. Cell growth is expressed by the increase in the square root of the measured area. This assay does not require isotopes and can be used to test tumor cells freshly dissociated from solid tissues. It permits cell-cell interactions which may change the sensitivity of tumor cells to various treatments. The extended period of observation allows the testing of multiple treatments. Surviving cells can also be recovered for further study. This assay may be useful for testing the efficacy of monoclonal antibody treatment on solid tumors.
Natural killer activity of lymphocytic infiltrates in mouse mammary lesions.
Tissue infiltrating lymphocytes were isolated from BALB/c line C4 preneoplastic hyperplastic alveolar nodules (HAN) and spontaneous tumours that arose from the HANs. NK activity of the lymphocytic infiltrates was tested in a 4 h chromium release assay using 51Cr labelled YAC cells. In situ lymphocytes of C4 HAN expressed 3-4 fold greater relative lytic activity (Pross et al., 1981) than did normal spleen cells whereas the relative lytic activity of C4 tumour infiltrates was equivalent or less than that of normal spleen cells. Spleen cells of all lesion bearers had reduced cytolytic capacity. YAC cell lysis by spleen cells and HAN infiltrates correlated with increasing E/T ratios. The degree of YAC lysis by C4 tumour infiltrates, however, either decreased, stayed the same, or increased non-exponentially with increasing E/T ratios especially at E/T greater than 50. Indeed, C4 tumour infiltrates from animals pretreated with anti-asialo GM1 (ASGM) could suppress the NK activity of normal spleen cells. The lytic activity of both C4 HAN and tumour infiltrates could be enhanced or depressed by in vivo treatment with poly IC or anti-ASGM, respectively. These results indicate that NK cells are activated or recruited into C4 preneoplastic lesions but their lytic activity wanes and suppressive activity arises with progression to neoplasia.