Effect of cytochalasin B on the maturation of enveloped viruses.

The maturation of two enveloped viruses, influenza and vesicular stomatitis, occurs in cells treated with cytochalasin B. Virions produced in the presence of 50 microgram/ml cytochalasin B (CB) appear to be as infectious as those from control cells, indicating that polymerized actin is not required for the assembly of functional viral components. CB inhibits the release of influenza virus from treated cells, a phenomenon which appears to be a result of the synthesis of an aberrant neuraminidase (NA) glycoprotein; virions grown in CB-treated cells had a 90% reduction in specific enzymatic activity. We found that both influenza viral glycoproteins (NA and Hemagglutinin glycoprotein) had faster electrophoretic mobilities and were more heterogeneous in CB-treated cells as compared with controls. We also observed complete inhibition of incorporation of labeled glucosamine into viral glycoproteins in the presence of the drug. It was of interest that CB-induced inhibition of glycosylation appeared to cause loss of neuraminidase function, whereas hemagglutinating activity was not noticeably impaired. The presence of altered glycoproteins did not significantly diminish the infectivity of either influenza virus or vesicular stomatitis virus. Our results indicate that no step in the maturation of enveloped viruses is dependent upon an intact cytoskeletal network.

Augmentation of macrophage complement receptor function in vitro. I. Characterization of the cellular interactions required for the generation of a T-lymphocyte product that enhances macrophage complement receptor function.

The function of complement receptors of mouse peritoneal macrophages was converted in vitro from mediating only attachment of macrophage complement receptor function was achieved by treating freshly explanted macrophages with supernates from cultures containing T lymphocytes and appropriately triggered macrophages. Fc receptor-mediated phagocyctosis by macrophages was required for the production of active supernates, for neither ingestion via the cells' complement receptors nor ingestion via nonimmunologic means was a sufficient stimulus for the macrophages' participation in the generation of supernatant activity. Fc receptor-triggered macrophages interacted by a contact dependent, but histocompatibility independent, mechanism with T lymphocytes, thereby signalling the lymphocytes to elaborate the active product. The possible significance of enhanced macrophage complement receptor function in inflammation, host defense against microbial pathogens, immune complex disease, and neoplasia is discussed.

VH gene expression is restricted in anti-IgG antibodies from MRL autoimmune mice.

Antibodies directed against IgG and DNA are found in the sera of autoimmune MRL/Mp lpr/lpr mice. Little is known of the molecular mechanisms underlying expression of such autoantibodies. We have investigated the binding diversity and pattern of VH gene expression in a panel of murine anti-IgG antibodies. We constructed eight hybridoma clones secreting IgM antibodies that bound to mouse IgG by using spleen cells from MRL/Mp lpr/lpr mice varying in age from 4 to 15 wk; one clone was derived from a 32-wk-old MRL +/+ mouse. The monoclonal IgM products exhibited varying binding specificities for intact mouse IgG, fragments of mouse IgG [Fc, Fab, (Fab')2], and heterologous IgG. Two of these antibodies crossreacted with B and/or Z DNA. Probes from seven of eight identified mouse VH gene families (7183, S107, Q52, J558, J606, 36-60, and 3609) were hybridized under high-stringency conditions with cytoplasmic RNA blots from each clone. Six clones hybridized only with the probe from the five-member 36-60 family. The remaining three clones crosshybridized with the 36-60 probe and the probe from the 60 member J558 family, perhaps reflecting somatic mutation from the original germline VH gene resulting in recognition by a probe from another family, in addition to the probe from the original germline family. Our results indicate that spontaneous MRL lpr/lpr anti-IgG antibodies are encoded predominantly by the 36-60 VH gene family and imply a nonrandom selection of this VH gene family in the production of these antibodies.

Diversity in the germline antibody repertoire. Molecular evolution of the T15 VN gene family.

The T15 heavy chain variable region (VH) gene family in BALB/c mice includes four elements each greater than 88% homologous with the other. One of these elements, V1, encodes virtually all of the VH regions in BALB/c antiphosphorylcholine antibodies, while another element, V3, is a pseudogene and cannot be transcribed or translated. We have examined the structural features of this VH gene family in other mouse strains and, in particular, have cloned and sequenced the alleles of these gene segments present in B10.P mice. Each of the four B10.P sequences can be matched with its allelic counterpart in BALB/c mice. This represents the first successful analysis of allelism in antibody variable region gene segments. The V1B10.P allele, like its BALB/c counterpart, encodes most of the known phosphorylcholine binding heavy chains from C37BL/6 mice. Similarly, the V3B10.P gene segment is a pseudogene like V3BALB, although only two of four abnormalities present in the BALB/c allele are also present in the B10.P allele. Careful analysis of the specific substitutions observed in the T15 VH gene family suggests that environmental selection for functional combining regions contributes significantly to the pattern of variation in the germline antibody repertoire. In addition, evidence is presented supporting frequent gene conversion events in the divergence of antibody genes.

Studies on the mechanism of phagocytosis. II. The interaction of macrophages with anti-immunoglobulin IgG-coated bone marrow-derived lymphocytes.

We have examined the effect of the distribution of anti-immunoglobulin IgG molecules on the surface of bone marrow-derived lymphocytes upon the interaction of these cells with macrophages. Lymphocytes which were diffusely coated with antibodies to surface immunoglogulin were ingested by macrophages. Lymphocytes which had the same number of anti-immunoglobulin IgG molecules redistributed to one pole of the surface bound to the macrophages' Fc receptors but were not ingested. These results confirm our previous hypothesis that ingestion of an immunologically coated particle requires the sequential, circumferential binding of specific receptors on the plasma membrane of a phagocytic cell to immunologic ligands distributed over the entire particle surface. Macrophages which had bound capped lymphocytes by the macrophages' Fc receptors removed the immune complex caps from the lymphocyte surface without destroying the lymphocytes. These lymphocytes remained attached to the macrophage surface. The finding that macrophages can phagocytize immune complexes from the surface of a cell without destroying the cell to which these complexes are attached may be important in understanding the effects of antigens and antibodies on cells participating in a humoral immune response, in identifying the mechanisms by which chronic viral infections are established, and in defining the roles of blocking antibodies in tumor immunity.

Studies on the mechanism of phagocytosis. I. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane.

These experiments were designed to evaluate the role of macrophage plasma membrane receptors for the third component of complement (C) and for the Fc portion of IgG in the ingestion phase of phagocytosis. Sheep erythrocyte (E) were coated with anti-E IgG [E(IgG)]; these E(IgG) were then attached to cultivated monolayers of mouse peritoneal macrophages under conditions which reversibly inhibit ingestion of E(IgG). The E(IgG)-macrophage complexes were further incubated under similar conditions with an antimacrophage IgG fraction which blocks Fc receptor-mediated ingestion but has no effect upon ingestion mediated by other phagocytic receptors. When these cultures were subsequently incubated under conditions optimal for particle ingestion, phagocytosis of the IgG-coated erythrocytes did not occur; the erythrocytes remained bound to the Fc receptors of the macrophage plasma membrane. To determine whether ligands must cover the entire surface of an attached particle to permit ingestion of that particle, C-coated E [E(IgM)C] were bound to the C receptors of thioglycollate-induced (activated) macrophages at 4 degrees C. E(IgM)C-macrophage complexes were then trypsinized at 4 degrees C, a procedure which resulted in cleavage of erythrocyte-bound C3b molecules to a form of C3 not recognized by the macrophage receptors for C3b. Under the conditions used, trypsin did not affect the attachment of E(IgM)C to the macrophage surface or the macrophage receptors for C3b. When these trypsin treated E(IgM)C-macrophage complexes were incubated at 37 degrees C, the bound E(IgM)C were not ingested; the erythrocytes remained attached to the macrophage plasma membrane via the macrophage's C receptors. These results indicate that attachment of a particle to specific receptors on the macrophage plasma membrane is not sufficient to trigger ingestion of that particle. Rather, ingestion requires the sequential, circumferential interaction of particle-bound ligands with specific plasma membrane receptors not involved in the initial attachment process.

Treatment of meningeal breast cancer xenografts in the rat using an anti-p185/HER2 antibody.

The metastatic spread of breast cancer to the leptomeninges (LM) is a painful, debilitating, and usually lethal condition. Current therapies are generally ineffective or extremely toxic. The current study evaluated monoclonal antibody therapy in an animal model of LM human breast cancer. Monoclonal antibody 4D5, which recognizes the extracellular domain of the HER2/neu receptor, was administered into the cerebrospinal fluid of athymic rats implanted with human breast cancer cell lines. Continuous intraventricular administration of 4D5 inhibited growth of SKBR3 cells that overexpress HER2/neu but not of MCF7 cells, which do not. Inhibition was dose-dependent, with higher doses of 4D5 producing an improved response. i.p. administration of cisplatin in addition to 4D5 did not improve results. Continuous administration of 4D5 into the lumbar, as opposed to the ventricular intrathecal space, was not therapeutically effective. Treatment with 4D5 did not result in outgrowth of cells lacking expression of the HER2/neu receptor. These results suggest that 4D5, administered regionally, may palliate LM metastases from HER2/neu-overexpressing breast carcinoma.

Periapical biopsy report: an analysis of over a 10-year period.

Biopsy reports of specimens from the apices of 1659 teeth physically present at the time the biopsy was performed were reviewed. The following information was recorded from each report: (a) source of specimen, (b) sex of patient, (c) age of patient, (d) location of tooth associated with lesion, and (e) diagnoses reported. Data were recorded and analyzed by chi-square analysis to note significant differences. This study found that 52% of the lesions were granulomas, 42% cysts, 2% periapical scars, and 4% other disorders. No differences were found between males and females in regard to age and location of lesions. Overall, the most common location for lesions was the maxillary anterior, followed by maxillary posterior, mandibular posterior, and finally the mandibular anterior jaw. This also was the case for all age ranges except 60 to 69 yr of age where lesions in the maxillary posterior area were most common. More granulomas were detected in all areas except in the mandibular posterior area where cysts were more common.

Challenges to participating in a lifestyle intervention program: the Native American Diabetes Project.

PURPOSE: This paper describes the factors that American Indian teachers in the Native American Diabetes Project (NADP) reported affected participation in the NADP lifestyle education sessions. METHODS: A postsession exit interview was conducted with each of the 7 mentors (teachers) of the NADP sessions. Interview questions addressed general perceptions of the sessions, factors that kept participants from coming to the sessions, and attitudes toward diabetes and persons with diabetes. Interviews were transcribed and responses reflecting factors related to participation were marked and organized into topic areas. RESULTS: Mentors reported a range of factors that affected participation in the sessions, such as conflicts with community activities and beliefs/attitudes about diabetes. The latter factor includes program knowledge, recruitment methods, attitudes toward the program, and beliefs about diabetes. CONCLUSIONS: Asking community members what factors they believe affect participation is an important component of increasing participation in community-based programs. Community members can provide a valuable personal perspective of actual and potential conflicts in the community.

Participant satisfaction with a culturally appropriate diabetes education program: the Native American Diabetes Project.

PURPOSE: The purpose of this paper is to report on participant satisfaction with the Native American Diabetes Project diabetes education program. METHODS: A questionnaire was designed to measure satisfaction among participants in the diabetes education program, which consisted of five sessions designed according to the Transtheoretical Model of Change and Social Action Theory with input from community members. Eight pueblo communities participated in the program. Sessions were taught by community mentors in three sites in New Mexico. One site taught sessions in a one-on-one format, and two sites taught sessions in a group format. RESULTS: The results showed that participant satisfaction did not vary based on session delivery type or by session site. Overall, participants responded positively to sessions designed according to Social Action Theory and with cultural competency. Retention rates for the sessions were 81% for group sessions and 91% for one-one-one sessions. CONCLUSIONS: Using a strong theoretical framework and community input to design diabetes education sessions may be important factors in participant satisfaction and retention in diabetes lifestyle education sessions.

Recombinant DNA--potential for gene therapy.

Certainly, if progress in recombinant DNA technology continues at its present rate, we have every reason to expect many more major breakthroughs in the diagnosis and treatment of human disease. Much of the progress from which we already benefit is not the increased understanding of just the molecular basis of genetic disease, but also of the molecular mechanisms of viral, bacterial, and parasite pathogenicity. Exploiting cloned antigens from pathogens to make vaccines is an ever expanding approach to preventive medicine. The other realm from which we already benefit is that in which we have bacteria produce large quantities of product from normal cloned genes, such as insulin, for treatment of patients deficient in that gene product. Although we cannot expect to eliminate some, or even treat all, genetic disease within the foreseeable future, it is quite clear that research in the area of genetic engineering has vast potential for improving the conditions of mankind.

A novel pre-B cell precursor: phenotypic characterization and differentiation induction by dendritic cell-T cell mixtures.

We have derived from spleens of nude mice early B lineage cell lines that are dependent upon factor(s) in supernatants of the WEHI-3B cell line. The cells are phenotypically at the pre-pre-B cell stage of differentiation. These cells are large and have basophilic granules in the cytoplasm. They are negative for cytoplasmic and surface immunoglobulin heavy or light chain, surface B220, surface Thy 1.2 and surface Ia but are positive for cytoplasmic B220. These cells at this early stage of maturation can be induced by mixtures of DC-T cells to express B220 on their surfaces and later to synthesize and ultimately to secrete immunoglobulin. These events are associated with morphologic changes in the cells to a lymphoblastoid appearance. Different pattern of immunoglobulin secretion were induced by DC-T from different tissues. The inductive event appears to occur during cell contact of pre-pre-B cells with the inducing DC-T cell mixture, but it does not appear to require IL-3. These data indicate that DC-T cell mixtures can provide signals for B cell differentiation at pre-pre-B cell stage as well as pre-B and mature B cell stage.

Relationship of physical symptoms and physical functioning to depression in patients with heart failure.

OBJECTIVE: The purpose of this study was to determine the relative contribution of physical symptoms and physical functioning to depression in adult patients with heart failure during hospitalization and the early postdischarge period. DESIGN: An exploratory, correlational longitudinal design was used. PATIENTS: The sample included 170 subjects with heart failure. RESULTS: Subjects' mean scores on the depression scale indicated that subjects were not depressed on average; however, 30% of the sample (n = 52) had scores indicative of clinical depression. Both physical symptoms (r = 0.48) and physical functioning (r = -0.32) were moderately correlated with depression. Physical symptoms contributed 13% uniquely to the variance in depression while physical functioning contributed only 2% uniquely to the variance in depression. Multiple regression analyses indicated that physical symptomatology is more closely related to depression than is physical functioning in adults with heart failure. CONCLUSIONS: This study showed the patients with heart failure who had increased physical symptoms and poorer physical functioning reported increased symptoms of depression. Physical symptoms explained a greater portion of the variance in depression than did physical functioning. Thus, it appears that patients with heart failure are affected emotionally by both their physical symptoms and their limitations in their physical functioning, but depression is more strongly related to having more physical symptoms than having greater limitations in physical functioning.

Treatment with targeted vesicular stomatitis virus generates therapeutic multifunctional anti-tumor memory CD4 T cells.

A generally applicable, easy-to-use method of focusing a patient's immune system to eradicate or prevent cancer has been elusive. We are attempting to develop a targeted virus to accomplish these aims. We previously created a recombinant replicating vesicular stomatitis virus (VSV) that preferentially infected Her2/neu expressing breast cancer cells and showed therapeutic efficacy in an implanted Balb/c mouse tumor model. The current work shows that this therapy generated therapeutic anti-tumor CD4 T cells against multiple tumor antigens. CD4 T cells transferred directly from cured donor mice could eradicate established tumors in host mice. T cells were transferred directly from donor mice and were not stimulated ex vivo. Both tumors that expressed Her2/neu and those that did not were cured by transferred T cells. Analysis of cytokines secreted by anti-tumor memory CD4 T cells displayed a multifunctional pattern with high levels of interferon-γ, interleukin (IL)-4 and IL-17. Anti-tumor memory CD4 T cells traveled to the mesenteric lymph nodes and were activated there. Treatment with targeted recombinant replicating VSV is a potent immune adjuvant that generates therapeutic, multifunctional anti-tumor memory CD4 T cells that recognize multiple tumor antigens. Immunity elicited by viral therapy is independent of host major histocompatibility complex or knowledge of tumor antigens. Virus-induced tumor immunity could have great benefit in the prevention and treatment of tumor metastases.