Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies.
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Ampicillin Resistance↗
Biomedical subjects
R E Goodman
Publications and source records attributed to R E Goodman.
A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.
Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.
Clinical Laboratory Techniques↗
DNA-liposome versus adenoviral mediated gene transfer of transforming growth factor beta1 in vascularized cardiac allografts: differential sensitivity of CD4+ and CD8+ T cells to transforming growth factor beta1.
We have developed a model of transforming growth factor (TGF)beta1 gene transfer into mouse vascularized cardiac allografts to study the use of gene transfer as an immunosuppressive therapy in transplantation. Donor hearts were perfused with either DNA-liposome complexes or adenoviral vectors that encode the active form of human TGFbeta1. DNA-liposome mediated transfection prolonged allograft survival in approximately two-thirds of transplant recipients, while adenoviral delivery of TGFbeta1 was not protective. Protective TGFbeta1 gene transfer was associated with reduced Th1 responses and an inhibition of the alloantibody isotype switch. The protective effects of TGFbeta1 gene transfer were overridden by exogenous interleukin-12 administration. Interestingly, alloreactive CD4+ and CD8+ cells exhibited distinct sensitivities to TGFbeta1 gene transfer: CD4+ Th1 function was abrogated by this modality, although CD8+ Th1 function was not. Transient depletion of recipient CD8+ cells markedly prolonged the survival of grafts transfected with either DNA-liposome complexes or adenoviral vectors. Transgene expression persisted for at least 60 days, and Th1 responses were not detectable until CD8+ T cells repopulated the periphery. However, long-term transfected allografts appeared to exhibit exacerbated fibrosis and neointimal development. These manifestations of chronic rejection were absent in long-term transfected isografts, suggesting that long-term expression of active TGFbeta1 alone is not sufficient to induce fibrosis of the grafts. Collectively, these data illustrate the utility of immunosuppressive gene therapy as a treatment for transplantation when combined with additional conditioning regimens. Further, they illustrate that alloreactive CD4+ and CD8+ cells may be differentially influenced by cytokine manipulation strategies.
Adenoviridae↗
Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.
Evidence derived from human and animal studies strongly supports the notion that dysfunctional alveolar epithelial cells (AECs) play a central role in determining the progression of inflammatory injury to pulmonary fibrosis. We formed the hypothesis that impaired production of the regulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured AECs plays a role in the development of pulmonary fibrosis. To test this hypothesis, we used the well-characterized model of bleomycin-induced pulmonary fibrosis in rats. GM-CSF mRNA is expressed at a constant high level in the lungs of untreated or saline-challenged animals. In contrast, there is a consistent reduction in expression of GM-CSF mRNA in the lung during the first week after bleomycin injury. Bleomycin-treated rats given neutralizing rabbit anti-rat GM-CSF IgG develop increased fibrosis. Type II AECs isolated from rats after bleomycin injury demonstrate diminished expression of GM-CSF mRNA immediately after isolation and in response to stimulation in vitro with endotoxin compared with that in normal type II cells. These data demonstrate a defect in the ability of type II epithelial cells from bleomycin-treated rats to express GM-CSF mRNA and a protective role for GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.
Animals↗
Induction of lung fibrosis in the mouse by intratracheal instillation of fluorescein isothiocyanate is not T-cell-dependent.
Pulmonary fibrosis is the pathological result of a diverse group of insults. Common features of this group of diseases include chronic inflammation and immune cell activation. The pathogenesis of pulmonary fibrosis is not well defined and the prognosis is poor, highlighting the need for good animal models to elucidate the cellular and molecular events that lead to pulmonary fibrosis. This paper provides insight on a newly described model of pulmonary fibrosis using a single intratracheal challenge with fluorescein isothiocyanate (FITC). Balb-c and C57BL6 mice given intratracheal FITC develop acute lung injury followed by chronic inflammation. Significant increases in lung collagen content compared to saline-treated mice are noted at day 21 after inoculation. T-cell-deficient animals develop similar increases in lung collagen content compared to immunocompetent controls despite the abrogation of specific anti-FITC serum antibodies. Thus, the induction of fibrosis in FITC-challenged mice is not dependent on T cell immunity. Persistent chronic inflammation and acute lung injury may be the inciting events for the development of lung fibrosis in this model.
Animals↗
Modulation of alloimmunity to major histocompatibility complex class I by cotransfer of cytokine genes in vivo.
Major histocompatibility complex (MHC) class I antigen is a potent stimulus for alloimmune responses and is the principal immunologic target mediating acute cellular rejection of allografts. Using a method of direct in vivo gene transfer of cDNA encoding donor type MHC class I, we showed in a rat model that recipient muscle could express the transferred MHC class I cDNA, resulting in alloimmunization of the recipient. This was most graphically demonstrated by accelerated rejection of cardiac allografts expressing the same MHC class I as encoded by the immunizing cDNA. We now report the use of the particle-mediated gene transfer via a gene gun (Geneva, Middleton, WI, USA) to transfer MHC class I, as well as cytokine gene expression vectors, into rat skin. Compared to intramuscular injection, gene gun transfer to skin resulted in more efficient immunization. Donor-specific cytotoxic T lymphocyte (CTL) responsiveness and antibody levels increased. Furthermore, coexpression of certain cytokine genes with the MHC class I cDNA modulated the immune response. Specifically, coimmunization with IL-10 cDNA abrogated immunity to allo-MHC class I, while coimmunization with GM-CSF cDNA enhanced it. The influence of expression of these genes in skin was demonstrated by alteration of donor cardiac allograft survival. This model is useful for induction and modulation of alloimmune responses and may be used to develop gene therapy strategies to modify them.
Animals↗
Products from plant biotechnology.
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Biotechnology↗
CD28 blockade alters cytokine mRNA profiles in cardiac transplantation.
BACKGROUND: T-cell response to alloantigen is dependent on T-cell receptor activation and costimulation through the CD28 receptor, because T-cell receptor activation alone is insufficient for optimal immune response. The CD28 receptor on helper T cells interacts with its ligand B7 on activated B cells-macrophages as costimulus to support T-cell activity. CTLA4Ig is a recombinant inhibitor of CD28 receptor activation. In vivo studies with a rat major histocompatibility complex mismatch heterotopic cardiac transplant model demonstrate that CTLA4Ig prolongs cardiac allograft survival. This CTLA4Ig survival benefit is enhanced with prior donor-specific antigen exposure. METHODS: To investigate CTLA4Ig mechanisms, we examined the differential expression of B7 and cytokine mRNAs for interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-10 (Th1 or Th2 activation) in cardiac allografts after treatment with CTLA4Ig and donor-specific antigen exposure versus conventional immunotherapy (cyclosporine, cyclophosphamide, or antilymphocyte serum). In the above major histocompatibility complex mismatch model, hearts (on day 5 after transplantation at peak rejection) had cytokine mRNA expression determined by semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: Inhibition of B7 expression was observed in CTLA4Ig animals. Expression of IL-2 and IFN-gamma was near undetectable in CTLA4Ig and cyclophosphamide rats but was only moderately reduced by cyclosporine and antilymphocyte serum. IL-4 mRNA expression was reduced equally in all animals. Finally, IL-10 levels were unchanged by CTLA4Ig but were decreased by other therapies. CONCLUSIONS: The beneficial effect of CTLA4Ig, inhibiting expression of B7, alters Th1 cytokines IL-2 and IFN-gamma, with a resultant predominant IL-10 driven, Th2 tolerogenic response.
Abatacept↗
Understanding human sexuality--specifically homosexuality and the paraphilias--in terms of chaos theory and fetal development.
This paper considers human sexual orientation, specifically homosexuality and some paraphilias, to occur as a result of intrauterine development, itself a mathematically chaotic process. Parameter space, an example of state space in the phase diagram, has been used to illustrate different phenotypes. The crossing of a bifurcation boundary by the developing fetus is proposed as a mechanism by which it may be changed from one sexual orientation to another, e.g. from heterosexuality to homosexuality. The factors which push the fetus over a bifurcation boundary, which include a Y-chromosome, specific hormone administration, the lying contiguous to an opposite-sex fetus in multiple pregnancies, maternal stress and immune factors are described. The syndromes congenital renal hyperplasia and the androgen insensitivity syndrome and their relevance to this model are also discussed. Finally, chaos theory is used to encompass the complex interactions between fetal development and cultural factors in human sexuality.
Bisexuality↗
IL-12 antagonism induces T helper 2 responses, yet exacerbates cardiac allograft rejection. Evidence against a dominant protective role for T helper 2 cytokines in alloimmunity.
IL-12 promotes Th1 development and inhibits the generation of Th2 by inducing IFN-gamma production. In several experimental models, Th2 are preferentially induced in the absence of IL-12. It was proposed that the preferential induction of Th2 by IL-12 antagonism would inhibit Th1-driven rejection responses, thereby promoting allograft acceptance. To test this possibility, mouse cardiac allograft recipients were treated with either anti-IL-12 Abs, or with the IL-12 receptor antagonist p40 homodimer. Unmodified rejection is characterized by a Th1-dominated response, with Th2 cytokines being absent or only weakly expressed within the allograft. Though both forms of IL-12 antagonism induced Th2 cytokine expression within the allograft, these treatments surprisingly exacerbated graft rejection relative to control animals. Interestingly, IL-12 antagonism did not inhibit IFN-gamma gene expression or in vivo sensitization of IFN-gamma-producing cells. Similar observations were made when IL-12 p40 knockout mice were used as allograft donors and recipients, verifying that IL-12 was not required for Th1 development. Further, IL-12 antagonism was associated with strong expression of p40 and weak expression of p35 within the graft. Neither p35 nor p40 mRNAs were detectable in control allografts. These data indicate that while IL-12 antagonism does induce Th2 cytokine expression within cardiac allografts, Th2 cytokines do not play a dominant protective role in the rejection process. Further, the Th2-inducing activity of IL-12 antagonism is not related to decreased IFN-gamma production, but may reflect altered regulation of IL-12 itself.
Animals↗
BUPA's discretionary recognition of treatment. Psychosexual medicine is no longer recognised for benefit purposes.
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Humans↗
Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumonia.
Effective host defense against bacterial infection is dependent upon the vigorous recruitment and activation of neutrophils and macrophages. We hypothesized that IL-10 is produced in the setting of bacterial pneumonia, and this cytokine may attenuate host defense by inhibiting the expression of important activating and chemotactic cytokines. CD-1 mice were challenged with either 30 microliters of saline or saline containing 10(3) CFUs of Klebsiella pneumoniae intratracheally (i.t.) and lungs were harvested at 8, 24, and 48 h. The i.t. inoculation with K. pneumoniae resulted in a 13-, 14-, and 8-fold increase in lung homogenate TNF, macrophage inflammatory protein-2 (MIP-2), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) levels, respectively, as compared with control animals. In addition, we observed an increase in IL-10 mRNA and protein levels in lung homogenates, maximal at 48 h postinoculation. To establish the biologic relevance of IL-10 in Klebsiella pneumonia, we passively immunized CD-1 mice with 0.5 ml of rabbit anti-murine IL-10 serum or preimmune serum i.p. 2 h before i.t. administration of K. pneumoniae. Treatment of animals with anti-IL-10 serum resulted in increased levels of TNF, MIP-2, and MIP-1 alpha, respectively, within lung homogenates at 24 and 48 h, as compared with preimmune-treated animals. Furthermore, neutralization of IL-10 resulted in a significant decrease in K. pneumoniae CFU in both lung homogenates and plasma harvested at 48 h, as well as a significant increase in survival in these animals. Our studies indicate that 1) IL-10 is produced during Klebsiella pneumonia; and 2) inhibition of IL-10 bioactivity in vivo results in enhanced bacterial clearance, increased expression of proinflammatory cytokines, and prolonged survival.
Animals↗
In vivo depletion of CD8+ T cells results in Th2 cytokine production and alternate mechanisms of allograft rejection.
A current hypothesis states that Th1 cytokines promote allograft rejection and that Th2 cytokines promote graft acceptance. We present data that question the tolerogenic activity of Th2 cytokines, and we suggest that Th2 cytokines may evoke allograft rejection by recruitment of alternate effector mechanisms. Unmodified rejection of mouse heterotopic cardiac allografts is associated with the accumulation of large numbers of donor-reactive CD8+ CTL within the allograft, which is indicative of a Th1-driven cellular response. However, when recipients are depleted of CD8+ CTL, rejection still occurs and is associated with an aggressive cellular infiltrate rich in eosinophils, large mononuclear cells, and fibroblast-like cells. Eosinophils, which are responsive to the Th2 cytokines IL-4 and IL-5, are not present in unmodified rejecting allografts. Differential production of Th1 versus Th2 cytokines was further suggested by altered levels of IgG2a (promoted by IFN gamma) and IgG1 (promoted by IL-4) alloantibody in the sera of these mice; IgG2a dominated the alloantibody response in unmodified allograft recipients, whereas IgG1 levels increased in recipients depleted of CD8+ CTL. Altered intragraft cytokine gene expression was verified by RT-PCR; Th1 (IL-2, IFN gamma), but not Th2 (IL-4, IL-5, IL-10), cytokine mRNAs were readily detectable in the allografts of unmodified recipients. In contrast, both Th1 and Th2 cytokine genes were expressed in the allografts of mice depleted of CD8+ CTL. These data suggest that donor-reactive CD8+ CTL inhibit intragraft production of Th2 cytokines, thereby promoting a Th1 dominated-rejection response. Elimination of CD8+ cells allows Th2 cytokine production, which may have deleterious, rather than protective, effects.
Animals↗
Interleukin 12: a potential link between nerve cells and the immune response in inflammatory disorders.
BACKGROUND: The nervous system has been implicated in several inflammatory skin disorders based on evidence such as the role of stress in inducing lesions, symmetry of lesions, and sparing of denervated skin. Interleukin 12 (IL-12) is a cytokine recently shown to promote cellular immune responses characterized by delayed-type hypersensitivity and production of the TH1-lymphokine, interferon-gamma. MATERIALS AND METHODS: Using immunohistochemistry, IL-12 immunoreactivity was identified in cryostat sections of normal and diseased human skin samples, and in the peripheral and central nervous system of rodents and human tissue samples. IL-12 p35 and p40 mRNAs were detected using reverse transcriptase-polymerase chain reaction in tissue samples and cultured cells. IL-12 protein levels were also examined by ELISA and quantitative bioassay utilizing an IL-12-dependent cell line. RESULTS: By immunostaining IL-12 was detected in free nerve ending in the epidermis of normal and diseased skin samples, and also in the dermal nerve fibers. Strong reactivity was detected in axonal processes and in various glial cell types. In addition, IL-12 protein and mRNA were contained within cutaneous peripheral nerves and spinal cord tissues, and functional levels of IL-12 were produced by cultured Schwann cells. CONCLUSIONS: It is likely that IL-12 is important in initiating or propagating selected inflammatory skin lesions and in determining the pattern of disease that will develop. The presence of IL-12 in neural tissue suggests a mechanism whereby the nervous system can modify or amplify cutaneous and perhaps other immune responses.
Animals↗
Keratinocyte-derived T cell costimulation induces preferential production of IL-2 and IL-4 but not IFN-gamma.
By using superantigens, we have found previously that keratinocytes activated by IFN-gamma could serve as accessory cells, providing costimulatory signals needed to induce T cell proliferation. Here, we compared the profile of cytokines produced by T cells stimulated in the presence of activated keratinocytes with the response seen using professional APCs. When keratinocytes are used as accessory cells there is a specific defect in T cell IFN-gamma production, whereas IL-2 and IL-4 are induced at levels comparable with those seen when professional APCs are used as accessory cells. Because keratinocytes express BB-1, a CD28-ligand distinct from B7-1 or B7-2 (which are found on professional APCs), we examined the possibility that the defect in IFN-gamma production might be a result of nonproductive CD28 engagement. However, even when the CD28 pathway is directly activated by a stimulatory mAb, there is no induction of IFN-gamma production in keratinocyte-supported cultures. In these same cultures IL-2 production is increased 10-fold, thus demonstrating a specific deficiency in the induction of IFN-gamma rather than a failure to respond to CD28 stimulation. Analysis by reverse transcriptase-PCR and ELISA for the inducible p40 chain of IL-12 reveals that keratinocytes produce little if any messenger RNA and no protein for IL-12 p40 compared with professional APCs. Addition of rIL-12 to keratinocyte-supported cultures restores IFN-gamma levels to those seen when professional APCs are present. Finally, when T cells are restimulated and analyzed at later time points (10 to 14 days) we find a refinement in cytokine profiles: T cells stimulated in the presence of professional APCs produced the Th1 cytokines IL-2 and IFN-gamma, whereas T cells stimulated in the presence of activated keratinocytes produced only the Th2 cytokine IL-4. The specific ability of keratinocytes to induce a Th2 response seems most closely linked to their absence of IL-12 production, and may be important in the maintenance of peripheral tolerance to self-Ags or in the immune response to exogenous Ags, pathogens, or haptens encountered in skin.
Antigen-Presenting Cells↗
Cytokine profile of protective anti-Trichinella spiralis CD4+ OX22- and non-protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL-4 alone does not determine protective capacity.
We analysed the cytokine profile of a T cell subset (CD4+ CD45 RC-) that confers protection against Trichinella spiralis infection in rats. These CD4+ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-gamma and functional cytokine secretion for IL-4, IL-5, IFN-gamma, TNF-alpha and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4+ CD45 RC+ or CD8+), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-gamma in the protective CD4+ CD45 RC- cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-gamma or TNF-alpha was secreted by the protective CD4+ CD45- RC- cells. Whereas the non-protective CD4+ CD45 RC+ cells secreted significant levels of IL-4, IFN-gamma, mast cell differentiating activity and TNF-alpha but little IL-5 activity. Non-protective CD8+ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-gamma secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.
Animals↗
Bovine mammary lactoferrin: implications from messenger ribonucleic acid (mRNA) sequence and regulation contrary to other milk proteins.
The regulation of bovine mammary lactoferrin, an important component of the antimicrobial defenses of the mammary gland, is poorly understood compared with the other milk proteins. The complete sequence for bovine lactoferrin mRNA shows it to be highly homologous to other lactoferrins and transferrins. However, regional differences in the deduced AA sequence of bovine lactoferrin compared with human lactoferrin and transferrin imply functional differences between them. Steady-state levels of bovine lactoferrin mRNA (by Northern blot) in the bovine mammary gland indicate that bovine lactoferrin expression is minimal in the developing and lactating gland but is strongly induced by mammary involution. The overall regulation of bovine lactoferrin in the mammary gland appears to be contrary to that of the other milk proteins. Features identified in the mRNA of bovine mammary lactoferrin may contribute to the differences in regulation between lactoferrin and other bovine milk proteins and to differences in concentrations of lactoferrin in milk across species. Lactoferrin secretion by bovine mammary cells grown in vitro does not appear to be dependent on prolactin and shows regulation by substratum, serum, and cell population to be different from that for casein. In contrast to casein, efficient secretion of lactoferrin from the cell does not require detachment of collagen substratum.
Amino Acid Sequence↗
Synthesis and characterization of rat interleukin-10 (IL-10) cDNA clones from the RNA of cultured OX8- OX22- thoracic duct T cells.
A cDNA of the complete coding region of rat IL-10 was cloned and sequenced using RNA isolated from a cultured population of thoracic duct T-lymphocytes obtained from Trichinella spiralis infected animals. The OX8- OX22-T-helper cells were stimulated in vitro with Concanavalin A for 24 hours prior to harvest. Reverse transcription of cellular RNA was primed with oligo-dT followed by amplification of IL-10 specific cDNA by polymerase chain reaction with synthetic oligo nucleotide primers chosen from two highly conserved regions of mouse and human IL-10. The sequence of the coding region of the amplified, cloned rat IL-10 cDNA is 90% identical to the mouse and 82% identical to the human IL-10 cDNA coding regions.
Amino Acid Sequence↗