Biomedical subjects

M J Newman

Publications and source records attributed to M J Newman.

At least 55 records · Page 3Linked to original sources

PubMed1993

Transforming growth factor beta and the cell surface in tumor progression.

Type 1 transforming growth beta (TGF-beta 1) is a multifunctional regulator of cellular differentiation, motility and growth. It is capable of inhibiting or stimulating these processes depending on cell type, cell density, culture conditions and TGF-beta 1 concentration. TGF-beta 1 regulates growth, in part, by inducing the expression and secretion of various types of collagen, which participate in the control of cell adhesion and migration, as well as growth. TGF-beta 1 also regulates cell growth by controlling the response to epidermal growth factor (EGF) and other growth factors, in ways that can either decrease or increase their growth-promoting effects. Alterations in both negative and positive growth responses to TGF-beta 1 play important roles in tumor progression. Loss of sensitivity to growth inhibition by TGF-beta 1 can occur as a result of decreased expression of collagen. Acquisition of sensitivity to growth stimulation, and autocrine transformation by TGF-beta 1, are associated with aberrant EGF receptor regulation. Aberrant growth factor receptor regulation by TGF-beta 1 may be mediated by a protein kinase C (PKC)-dependent pathway which inhibits degradation of growth factor receptor/ligand complexes. The evidence reviewed is consistent with a minimal two-step mechanism for autocrine transformation, which involves production of growth factor and enhanced cellular response as a result of aberrant membrane traffic. Defects in membrane traffic regulation may provide an explanation for common alterations in tumor cell response to both multiple growth inhibitors and growth stimulators, and may also suggest novel approaches to cancer chemotherapy.

Animals↗

PubMed1993

Cryopreservation of bovine buffy coat leukocytes for use in immunologic studies.

A simple cryogenic technique for preserving bovine buffy coat leukocytes was developed. This was coupled with a variation of the standard discontinuous gradient technique to purify mononuclear cells that retained immunologic function. The total number of mononuclear cells recovered from cyropreserved samples were only 87 to 42% of those recovered from freshly obtained blood samples. However, the functional capabilities of mononuclear cells from cyopreserved buffy coat preparations were retained. Polyclonal proliferative responses to 3 mitogens were measured, using a titration of mitogen concentrations, and were found to be normal, compared with those of cells isolated from fresh blood. Blood samples collected after vaccination with Brucella abortus contained leukocytes that responded to irradiated B abortus. These antigen-specific responses were also retained through cyopreservation. Cell surface expression of T-lymphocyte antigens, CD2, CD4, and CD8, and cell-surface IgM on B lymphocytes was also evaluated. Flow cytometric analysis of fresh and cryopreserved mononuclear cell preparations indicated that the relative proportions of different subpopulations were not altered. The technical simplicity of our cryopreservation system will allow processing of large numbers of samples with the ability to assay various immune functions at a later time.

Animals↗

PubMed1992

Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T lymphocyte responses.

The ability of a saponin adjuvant, QS-21, to induce OVA-specific, class I MHC Ag-restricted CTL was investigated using different forms of soluble OVA and OVA adsorbed onto alum as immunogens. C57BL/6 mice were immunized with soluble native or denatured OVA in formulations that contained increasing quantities of QS-21, and CTL responses were measured using EL4 and E.G7-OVA cells as targets and splenic mononuclear cells as effectors. Ag-specific CTL responses were produced but only if the QS-21 adjuvant was used. Similar responses were induced using alum-adsorbed OVA when mixed with the QS-21 adjuvant but not when used alone. The CTL were specific for an epitope present on the OVA258-276 synthetic peptide, which contains the dominant CTL epitope recognized by C57BL/6 mice. The CD8+ subpopulation of lymphocytes in immune mice was not increased in spleens but increased significantly in vitro after culture with soluble OVA. The CTL activity of splenic mononuclear cell preparations was totally destroyed by treatment with mAb specific to the CD8 Ag plus complement. The ability of the QS-21 adjuvant to induce class I MHC Ag-restricted CTL after immunization with soluble proteins is a characteristic unique to saponin adjuvants.

Adjuvants, Immunologic↗

PubMed1992

Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine.

The adjuvant activity of a single highly purified saponin from the soap bark tree Quillaja saponaria was evaluated by using it as a component in an experimental vaccine containing rHIV-1 envelope protein (HIV-1 160D) adsorbed to alum. BALB/c mice immunized with experimental vaccine formulations containing the saponin adjuvant QS-21 produced significantly higher titers of antibodies than mice vaccinated with only the alum-adsorbed HIV-1 160D. Potent amnestic antibody responses to HIV-1 viral proteins were also induced. Ag-specific proliferative responses to recombinant proteins and to three variants of HIV-1 were significantly increased using QS-21 as an adjuvant. Alum-adsorbed HIV-1 160D failed to induce measurable proliferative responses to inactivated HIV-1 viruses, but group-specific proliferative responses were raised when the QS-21 adjuvant was used in the vaccine formulation. MHC class I restricted CTL specific for the immunodominant V-3 loop were induced but only when the QS-21 adjuvant was included in the vaccine formulation. The production of serine esterase by Ag-activated splenic mononuclear cells, indicating the maturation of precursor CTL, was used as a secondary measure of CTL activity, and this response was also increased. The specificity of antibody responses was not significantly broadened using QS-21; the adjuvant increased the immune recognition of epitopes throughout the HIV-1 glycoprotein 160. However, the specificity of the proliferation and serine esterase responses was broadened, suggesting that the QS-21 augmented cell-mediated immune responses specific for epitopes outside of the V-3 loop. Additionally, the QS-21 adjuvant appeared to induce recognition of weakly immunogenic epitopes that were not recognized using only alum-adsorbed HIV-1 160D. The ability of QS-21 to augment both antibody and cell-mediated immune responses suggests that this adjuvant could be a valuable component in subunit vaccines.

AIDS Vaccines↗

PubMed1992

Preliminary report on the isolation of a dysgonic variety of Microsporum canis together with the normal variety from a cattery.

The isolation of a dysgonic variety of Microsporum canis from a large number of cats and kittens in a cattery is described. The normal variety of this fungus was isolated at the same time from the same animals. Dysgonic varieties are thought to be mutants of normal strains, but this isolation of both forms together suggests that the relationship may be more complex.

Animals↗

PubMed1992

Immunogenicity and toxicity testing of an experimental HIV-1 vaccine in nonhuman primates.

A highly purified saponin from Q. saponaria (QS-21) was tested in juvenile rhesus macaques for adjuvant activity and toxicity. The QS-21 was tested alone or as part of an experimental subunit HIV-1 vaccine containing a truncated recombinant HIV-1 envelope protein (gp160D) adsorbed to alum. Antibody responses were measured using ELISA and cell-mediated immunity was measured using cellular proliferation assays. Potential toxicity was monitored by standard clinical pathology testing using peripheral blood and urine samples. No toxic effects were observed, even after the administration of the experimental vaccines three times at monthly intervals. The QS-21 saponin adjuvant enhanced total antibody production levels by greater than 100-fold and broadened the specificity of the response so that additional epitopes were recognized, when compared with alum-adsorbed HIV-1 gp160D formulation. Low-level, antigen-specific proliferative responses to HIV-1 recombinant gp160 were induced by either vaccine formulation. Proliferative responses were induced by a sham challenge with soluble recombinant HIV-1 gp160 for all of the animals that had been vaccinated. However, those that received the HIV-complete vaccine formulation containing QS-21 responded significantly better. These data demonstrated that the QS-21 adjuvant augmented both antibody responses and cell-mediated immunity and established immunological memory. The potent adjuvant activity and lack of toxicity suggest that this adjuvant should be safe and effective for use in HIV-1 vaccines.

AIDS Vaccines↗

PubMed1991

In vitro measurement of antigen-specific cell-mediated immune responses using recombinant HIV-1 proteins adsorbed to latex microspheres.

Recombinant proteins representing full-length and truncated forms of the human immunodeficiency virus type 1 envelope protein gp160 were produced in E. coli and sf9 insect cells. These proteins were denatured and reduced as a function of purification. We adsorbed these proteins onto latex microspheres and used the protein-coated particles as a vehicle to present the antigen in vitro to splenic mononuclear cells from immune mice. Recombinant proteins presented on the latex particles induced antigen-specific proliferative responses that were dependent on the antigen concentration. The proliferative responses were similar to those produced against an identical protein used in soluble form and equivalent protein concentrations. Latex microspheres coated with recombinant proteins could also induce precursor cytotoxic T lymphocytes to mature to functional effector cells in vitro. The use of the latex microspheres to present recombinant proteins as antigens allowed for the use of denatured proteins in our assay that were not soluble in aqueous solutions, such as cell culture media. This system of delivering recombinant proteins in vitro should greatly facilitate the use of recombinant proteins in assays involving live cells.

Animals↗

PubMed1991

Transient suppression of equine immune responses by equine infectious anemia virus (EIAV).

Suppression of the immune system is a common aspect of the disease pathogenesis associated with retroviral infections in both man and animals. We have measured transient suppression of the equine immune system as a loss or decrease in antigen-specific and polyclonal lymphocyte proliferation following experimental infection of ponies with three variants of equine infectious anemia virus (EIAV) with difference virulence characteristics. The transient suppression of proliferative responses was temporally associated with recurrent febrile episodes, which are the hallmark symptom of EIAV-induced disease. Decreased proliferative responses occurred at all times when EIAV viremia was identified, based on the detection of an infectious virus in plasma or viral proteins on peripheral blood mononuclear cells. The immunosuppression was observed most frequently in ponies infected with virulent variants of EIAV which suggested that this effect may contribute to disease pathogenesis. Suppression of polyclonal proliferative responses was induced in vitro by the addition of either infectious or heat-inactivated EIAV to cultures, demonstrating that the viral structural proteins were immunosuppressive in the absence of infection. These studies indicated that EIAV is similar to other retroviruses in that it has the ability to suppress the immune system.

Animals↗

PubMed1990

Evaluation of the role of extracellular matrix proteins, polyunsaturated fatty acids and c-myc expression in the inhibition of the serum-free growth of epithelial cells by TGF-beta 1.

Novel or modified serum-free media were developed for the anchorage-dependent growth of nontransformed murine mammary epithelial cells (MMEC) and Balb/MK murine keratinocytes respectively. Growth rates for both cell lines were similar in serum-containing and serum-free media. The serum-free media were used to evaluate potential mechanisms of epithelial cell growth regulation by type 1 transforming growth factor beta (TGF-beta 1). The growth of MMEC and Balb/MK cells was reversibly inhibited 40-65% in a time- and dose-dependent fashion by TGF-beta 1 under both serum-containing and serum-free conditions. Constitutive over-expression of a transfected c-myc oncogene in MMEC did not result in loss of sensitivity to growth inhibition by TGF-beta 1. In addition, Balb/MK and MMEC growth inhibition by TGF-beta 1 was not potentiated by polyunsaturated fatty acids or reversed by vitamin E. Exogenous type V collagen was able to mimic the inhibitory effects of TGF-beta 1 on the serum-free growth of Balb/MK and MMEC. In contrast, collagen types I and IV, fibronectin and laminin did not inhibit the growth of these cells. The type V collagen used was not contaminated with TGF-beta, and subsaturating, but not saturating concentrations of type V collagen and TGF-beta 1 were additive with respect to Balb/MK and MMEC growth inhibition. These results demonstrate that nontransformed epithelial cell growth inhibition by TGF-beta 1 is mediated by mechanisms distinct from those observed with certain carcinoma and melanoma cells. Our results also suggest the possible involvement of type V collagen in Balb/MK and MMEC growth inhibition by TGF-beta 1.

Animals↗

PubMed1990

Antibodies to the neural cell adhesion molecule disrupt functional recovery in injured nerves.

Tubes containing specific monoclonal antibodies to the neural cell adhesion molecule (N-CAM) were applied to transected sciatic nerves to attempt to perturb the recovery of muscle function. Physiological recordings were used to estimate the return of function. The decline of implanted antibody over 28 days was estimated and negatively correlated with the degree of functional recovery. No significant immune responses were detected in response to the implanted material. The data implicated N-CAM as a significant component of nerve regeneration.

Animals↗

PubMed1990

Cryopreservation of equine mononuclear cells for immunological studies.

A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples by measuring the ability of cells to proliferate in response to mitogens and specific antigens. Cell-surface antigen expression was measured using monoclonal antibodies in conjunction with flow cytometric techniques and alloantisera in a complement mediated cytotoxicity assay. Cryopreserved mononuclear cells were capable of proliferating normally when stimulated with several mitogens, pokeweed mitogen, phytohemagglutinin and concanavalin A, and a single specific antigen preparation, equine influenza-2 (Equi-2) proteins. The maximum levels of proliferation induced by varying the concentrations of mitogens or the Equi-2 proteins were the same for both the fresh and cryopreserved cells. However, the cryopreserved cells usually required one more day in culture to attain maximum proliferation levels. Flow cytometric analysis of the samples demonstrated that the relative proportions of different lymphocyte populations were not altered by the cryopreservation step. Similarly, MHS alloantigen expression was not altered. The simplicity of the technique coupled with the retained functional properties allows for the cryopreservation of large numbers of leukocytes and the ability to assay various immune functions at a later time.

Animals↗

PubMed1990

Inhibition of carcinoma and melanoma cell growth by type 1 transforming growth factor beta is dependent on the presence of polyunsaturated fatty acids.

Improved serum-free media were developed for the anchorage-dependent growth of A549 human lung carcinoma and B16 mouse melanoma cell lines in vitro. Type 1 transforming growth factor beta (TGF-beta 1) inhibited the growth of A549 or B16 cells under serum-free conditions or in the presence of 10% serum by 15-33%. In contrast, in the presence of micrograms/ml concentrations of polyunsaturated fatty acids (PUFAs), picomolar concentrations of TGF-beta 1 irreversibly inhibited the serum-free growth of A549 or B16 cells by 90-100%. The PUFAs alone had little effect on cell growth. Cell growth inhibition by TGF-beta 1 was not potentiated by saturated fatty acids, monounsaturated fatty acids, or prostaglandins. Inhibition of A549 or B16 cell growth by TGF-beta 1 in the presence of PUFAs was almost completely reversed by the antioxidant vitamin E, suggesting a role for lipid peroxidation in this process. Inhibition of A549 or B16 cell growth by TGF-beta 1 in the presence of 5% fetal calf serum was also potentiated by PUFAs and partially reversed by antioxidants. The presence of retinoic acid was required for maximal PUFA-dependent growth inhibition of A549 or B16 cells by TGF-beta 1 under some, but not all, conditions. These results suggest that inhibition of carcinoma and melanoma cell growth by TGF-beta 1 is mediated, in large part, by PUFAs.

Animals↗

PubMed1990

Serological characterization of polycyclic aromatic hydrocarbon diolepoxide-DNA adducts using monoclonal antibodies.

Polycyclic aromatic hydrocarbons (PAHs) are a group of structurally related compounds that are present in the environment in complex mixtures as common pollutants. These compounds have been studied extensively because of their carcinogenic and toxic properties to humans. We reported previously that humans exposed to certain PAHs produce antibodies that bind to different PAH diolepoxide-DNA (PAH-DNA) adducts. The ability to detect and measure antibodies to PAH-DNA adducts in human blood samples could prove useful as a biological dosimeter for identifying persons that have been exposed to high levels of PAHs, i.e. persons who may be at high cancer risk. In our initial studies we found that it was common for persons who were exposed to PAH to produce antibodies against PAH-DNA adducts. However, we were unable to identify the actual chemical types of PAH-DNA adducts that were recognized by the serum antibodies because many serum samples contained antibody activity to more than one adduct. These data indicate that different PAH-DNA adducts may be serologically similar or that humans actually produce immune responses against more than a single PAH-DNA adduct. We have used monoclonal antibody technology to determine the extent to which different PAH-DNA adducts share serologically recognized epitopes. Monoclonal antibodies were produced against two different PAH-DNA adducts, benzo[a]pyrene diolepoxide-DNA (BPDE-DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA). The binding of these antibodies to five PAH-DNA adduct preparations and to soluble PAHs was assessed. We found that most monoclonal antibodies bound to more than a single type of PAH-DNA adduct, documenting the serological relatedness of different PAH-DNA adducts. However, two monoclonal antibodies were produced that bound only to BPDE-DNA. Soluble non-metabolized PAHs and PAH tetraols were not recognized by these antibodies, thus demonstrating their specificity for PAH-DNA adducts and not the PAHs alone. Monoclonal antibodies produced against BADE-DNA also bound to chrysene diolepoxide-DNA but not to BPDE-DNA or to two other PAH-DNA adducts. These data demonstrate that significant serological cross-reactivity exists between different PAH-DNA adducts and support our contention that human antibodies to PAH-DNA can often bind to more than a single type of PAH-DNA adduct. However, our ability to produce monoclonal antibodies with restricted specificity suggest that the production of highly monoclonal antibody reagents for use in assays designed to measure PAH-DNA adducts in biological samples is feasible.

Animals↗

PubMed1990

Molecular mechanics and antibody binding in the structural analysis of polycyclic aromatic hydrocarbon-diol-epoxide--DNA adducts.

Analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts using monoclonal antibodies raised against DNA that had been modified with (+-)-r-7-,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in an enzyme-linked immunosorbent assay, as well as analysis using human serum antibodies and antibodies raised in laboratory animals, have suggested the presence on these adducts of both common and unique immunological epitopes. The molecular mechanics studies reported here establish a model for the analysis of PAH-DNA adducts through the identification of energetically favored binding conformations and they further reveal structural alterations in DNA due to the presence of carcinogen adducts. The data explain the antibody reactivity patterns by defining different molecular presenting surfaces that are available for antibody binding. The preferred orientation of the aromatic portions of the adducts, which align either 3' or 5' in the minor groove, were found to be correlated with antibody reactivity patterns. Examination of the topographical characteristics of the adducts facilitated correlation of adduct-antibody recognition and adduct presenting surface. Significant differences were found between benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts, which align 5' in the minor groove, and benz[a]anthracene-diol-epoxide (BADE)-DNA and dibenz[a,c]anthracene-diol-epoxide-DNA adducts, which align 3' within the minor groove. Chrysene-diol-epoxide-DNA adducts were found to have only a weak preference for 5' alignment and therefore share topographical characteristics with both BPDE-DNA and BADE-DNA adducts.

Animals↗

PubMed1990

Characterization and purification of a secreted plasminogen activator inhibitor (PAI-1) induced by transforming growth factor-beta 1 in normal rat kidney (NRK) cells: decreased PAI-1 expression in transformed NRK cells.

Type 1 transforming growth factor-beta (TGF beta 1) was found to be a potent inducer of the secretion of a 49,000 mol wt protein by normal rat kidney (NRK) cells. This protein was related to type 1 plasminogen activator inhibitor (PAI-1) on the basis of molecular mass, activity in the presence of sodium dodecyl sulfate, immunoprecipitation by antibodies to PAI-1, and N-terminal sequence analysis. PAI-1 levels in the conditioned medium of NRK cells were increased 5- to 11-fold when cells were incubated with picomolar concentrations of TGF beta 1 for 24 h, reaching a concentration of approximately 0.3 microgram/ml. The secreted PAI-1 was deposited in the NRK extracellular matrix as well as released into the culture medium. A spontaneously transformed NRK cell line was found to secrete 3-4 times less PAI-1, in the absence or presence of TGF beta 1, compared to the parent cell line, while PAI-1 secretion in Kirsten sarcoma virus-transformed NRK cells was almost completely abrogated. A novel purification procedure was established, which results in the isolation of highly active and detergent-free TGF beta 1-induced PAI-1.

Amino Acid Sequence↗

PubMed1989

Inhibition of normal rat kidney cell growth by transforming growth factor-beta is mediated by collagen.

We have investigated the mechanism of inhibition of the serum-free monolayer growth of normal rat kidney (NRK) cells by transforming growth factor-beta (TGF-beta). NRK cells grown on fibronectin-coated dishes exhibited a biphasic response to TGF-beta. Monolayer growth was slightly stimulated by subpicomolar concentrations, while picomolar concentrations of TGF-beta inhibited NRK cell growth in the presence or absence of epidermal growth factor. NRK cells exhibited a similar biphasic growth response to exogenous type I collagen. TGF-beta induced a 3-5-fold increase in the deposition of type I collagen-like proteins into the extracellular matrix of NRK cells during serum-free growth. Type I collagen-like proteins were identified by their sensitivity to degradation by purified bacterial collagenase and by Western blot analysis. The TGF-beta dose-response curves for induction of extracellular matrix-localized collagen and inhibition of NRK cell growth were similar. Finally, the inclusion of a purified bacterial collagenase, which did not degrade TGF-beta or TGF-beta receptors, or alter control NRK growth, prevented exogenous collagen or TGF-beta from inhibiting the serum-free growth of NRK cells. Our results demonstrate that an increase in collagen secretion plays an important role in the inhibition of the growth of NRK cells by TGF-beta.

Animals↗

PubMed1989

Growth stimulation, altered regulation of epidermal growth factor receptors, and autocrine transformation of spontaneously transformed normal rat kidney cells by transforming growth factor beta.

The tumorigenic NRK-PT14 cell line requires exogenous epidermal growth factor (EGF), but has lost the requirement for transforming growth factor beta (TGF-beta) for anchorage-independent growth, compared to normal rat kidney (NRK) cells. Development of an optimized serum-free medium for the growth of these cells revealed that NRK-PT14 cells also exhibit a qualitatively altered sensitivity to exogenous type 1 TGF-beta, compared to NRK cells. EGF-induced serum-free monolayer growth of NRK-PT14 cells was stimulated 2-fold by TGF-beta under conditions where growth of NRK cells was inhibited by 67%. TGF-beta only stimulated the growth of NRK-PT14 cells when EGF was present and when EGF was added before TGF-beta. In addition, the stimulation of EGF-induced NRK-PT14 cell growth by TGF-beta was associated with a specific, reversible loss of the high-affinity subpopulation of EGF receptors from the surface of these cells. Treatment of NRK cells with TGF-beta resulted in an increase in this EGF receptor population. Finally, EGF-induced anchorage-independent growth of NRK-PT14 cells was shown to be dependent on secreted TGF-beta, demonstrating an autocrine role for TGF-beta in the transformed phenotype of these cells. Autocrine transformation of NRK-PT14 cells by TGF-beta may result directly from the acquisition of an altered (positive) sensitivity to this growth factor.

Animals↗

PubMed1988

BoLA antigens are associated with increased frequency of persistent lymphocytosis in bovine leukaemia virus infected cattle and with increased incidence of antibodies to bovine leukaemia virus.

The association between bovine major histocompatibility system (BoLA) type and persistent lymphocytosis in cattle with antibodies to bovine leukaemia virus was examined by comparing antigen frequencies in cattle with persistent lymphocytosis to controls matched for age, sex, breed and presence of antibodies to BLV. The cattle came from nine dairy herds in south-east Queensland, Australia; six herds were Australian Illawarra Shorthorn (AIS), two herds were Jersey and one herd was Friesian. Antigen W6 and Eu28R were more common in cattle with persistent lymphocytosis than in controls. Antigen W8 was less common in AIS cattle with persistent lymphocytosis. A study of 24 offspring from one sire, heterozygous for W10 and Eu28R, showed that offspring inheriting Eu28R from the sire were significantly more likely to have antibodies to BLV than offspring inheriting the opposing W10 haplotype.

Animals↗