TRIO Method for Detection of Beta-Lactams, Sulfonamides, and Tetracyclines in Raw Commingled Cows' Milk.

A qualitative 3 min one-step assay for detecting beta-lactam, sulfonamide, and tetracycline antibiotics was validated following milk screening test guidelines developed by FDA-CVM, AOAC-RI, and IDF. The validated 90% detection levels with 95% confidence were: penicillin G 2 part per billion (ppb); amoxicillin 4 ppb; ampicillin 9 ppb; ceftiofur plus metabolites 50 ppb; cloxacillin 9 ppb; cephapirin 15 ppb; sulfadimethoxine 8 ppb; sulfamethazine 9 ppb; chlortetracycline 34 ppb; oxytetracycline 53 ppb; and tetracycline 42 ppb. Detection levels were lower than U.S. and Canadian allowable limits for milk and were consistent with most European Maximum Residue Limits. Tests of raw commingled cows' milk indicated a low positive error rate of <0.3% with no interferences demonstrated by 1.08 MM/mL somatic cells, Gram-positive or Gram-negative bacteria < 300 K/mL, freeze/thawing, or non-targeted drugs. Detection of incurred residues were similar to, or more sensitive to, fortified samples. Some cross reactivity across drug families occurred in interference studies and therefore initial positive samples should be confirmed with drug family specific screening methods. The National Conference of Interstate Milk Shipments approval as a bulk tank/tanker screening test was completed in three stages for each drug family, including a tetracycline confirmation procedure to target U.S. tolerance levels. Detection and robustness were found to be appropriate for multiple countries' regulatory requirements for screening tests. The method development, validation, and approval was intended to diversify and increase the verification tools for the control of the major antibiotic drug families used in managing cows' health and welfare.

The p53-independent tumoricidal activity of an adenoviral vector encoding a p27-p16 fusion tumor suppressor gene.

We describe here that DE1-adenovirus vectors (AV) expressing a p27-p16 fusion molecule, termed W9, induce tumor cell apoptosis when overexpressed in a wide range of tumor cell types. However, in primary human cells derived from a variety of normal tissues, AV-W9 induced minimal apoptosis. In tumor cells AV-W9 demonstrated 5- to 50-fold greater tumoricidal activity than either of the parental molecules p16 and p27. In these studies, AV-W9 elicited apoptosis independent of the p53 and Rb status of the tumor cells. In several murine tumor models AV-W9 demonstrated p53-independent antitumor activity. It completely prevented tumor formation in two ex vivo models, whereas the parental molecules resulted in partial protection. Furthermore, AV-W9 induced tumor regression or suppressed tumor growth when introduced intratumorally into preestablished tumors in mice. This effect may be mediated through tumor cell apoptosis or antiangiogenic activity of AV-W9. Thus, this novel chimeric molecule is more potent and capable of killing a broader spectrum of tumors than the parental p16 and p27 molecules independent of the tumor cell p53 and phenotype and represents a powerful new therapeutic agent for cancer gene therapy.

Antigen-specific cytolysis by neutrophils and NK cells expressing chimeric immune receptors bearing zeta or gamma signaling domains.

TCR- and IgG-binding Fc receptors (Fc gamma R) mediate a variety of critical biologic activities including cytolysis via the structurally related zeta- and gamma-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR zeta-chain. Such zeta-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both zeta- and gamma-CIRs to activate the cytolytic function of two distinct classes of Fc gamma R-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing zeta- and gamma-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both zeta- and gamma-based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the zeta-CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells.

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor zeta chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.

Chimeric zeta-receptors direct human natural killer (NK) effector function to permit killing of NK-resistant tumor cells and HIV-infected T lymphocytes.

Chimeric receptors in which a signaling component of the TCR complex such as zeta is fused directly to the ligand binding domain of a heterologous receptor or Ab have been shown to redirect the specific effector activity of T lymphocytes. We previously described the ability of two classes of such chimeric zeta-receptors bearing extracellular domains derived from either the HIV receptor CD4 (CD4 zeta) or an HIV-specific single chain Ab to redirect primary human CD8+ T cells to kill HIV-infected T cells. In this report we demonstrate that human NK cells can be genetically modified to express high levels of CD4 zeta using retroviral transduction. The CD4 zeta chimeric receptor is biochemically active, as cross-linking of CD4 zeta on NK cells results in tyrosine phosphorylation of CD4 zeta and multiple cellular proteins. More importantly, the CD4 zeta chimeric receptor is functionally active and can direct NK cells to specifically and efficiently lyse either NK-resistant tumor cells expressing the relevant ligand, gp120, or CD4+ T cells infected with HIV. These results show that human NK cells can be readily activated via zeta-based chimeric receptors to target both tumor and virally infected cells, and suggest a novel approach to the treatment of disease.

Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors.

We have developed an immunotherapeutic approach with potential application in the treatment of viral and malignant disease. We show that primary CD8+ T cells isolated from peripheral blood can be genetically modified by retroviral transduction to express high levels of universal (major histocompatibility complex-unrestricted) chimeric T-cell receptors specific for human immunodeficiency virus (HIV) antigens. Two classes of HIV-specific URs in which the antigen-binding domain is comprised of either CD4 or a single-chain antibody are capable of activating a number of T-cell effector functions in response to target cells, including cytolysis, in a highly sensitive and specific manner. Importantly, we have addressed a number of issues which, although particularly relevant to the clinical application of this approach in the treatment of HIV infection, may also impact on the potential of UR immunotherapy for other disease targets. The UR immunotherapeutic system is particularly suited for evaluation in the clinical setting.

Molecular regulation of the IL-2 gene: rheostatic control of the immune system.

The delivery of costimulation and the effects of the anergic state impinge on IL-2 production via different molecular mechanisms. The strongest experimental support at this stage suggests that CD28 signaling effects mRNA stability of several lymphokine genes including IL-2. While there may also be transcriptional effects of CD28 signals in human cells, controversy surrounding relevant TCR mimics must be addressed. In the case of clonal anergy, however, transcriptional non-responsiveness is evident when anergic cells are restimulated with TCR and costimulatory signals. This repression affects predominantly AP-1 activity. So far, the nature of the repression has not been identified.

Transactivation by AP-1 is a molecular target of T cell clonal anergy.

Anergy is a mechanism of T lymphocyte tolerance induced by antigen receptor stimulation in the absence of co-stimulation. Anergic T cells were shown to have a defect in antigen-induced transcription of the interleukin-2 gene. Analysis of the promoter indicated that the transcription factor AP-1 and its corresponding cis element were specifically down-regulated. Exposure of anergic T cells to interleukin-2 restored both antigen responsiveness and activity of the AP-1 element.

Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate antigen.

Oct-2 is a transcription factor that binds specifically to octamer DNA motifs in the promoters of immunoglobulin and interleukin-2 genes. All tumor cell lines from the B-cell lineage and a few from the T-cell lineage express Oct-2. To address the role of Oct-2 in the T-cell lineage, we studied the expression of Oct-2 mRNA and protein in nontransformed human and mouse T cells. Oct-2 was found in CD4+ and CD8+ T cells prepared from human peripheral blood and in mouse lymph node T cells. In a T-cell clone specific for pigeon cytochrome c in the context of I-Ek, Oct-2 was induced by antigen stimulation, with the increase in Oct-2 protein seen first at 3 h after activation and continuing for at least 24 h. Oct-2 mRNA induction during antigen-driven T-cell activation was blocked by cyclosporin A, as well as by protein synthesis inhibitors. These results suggest that Oct-2 participates in transcriptional regulation during T-cell activation. The relatively delayed kinetics of Oct-2 induction suggests that Oct-2 mediates the changes in gene expression which occur many hours or days following antigen stimulation of T lymphocytes.

NF-kappa B subunit regulation in nontransformed CD4+ T lymphocytes.

Regulation of interleukin-2 (IL-2) gene expression by the p50 and p65 subunits of the DNA binding protein NF-kappa B was studied in nontransformed CD4+ T lymphocyte clones. A homodimeric complex of the NF-kappa B p50 subunit was found in resting T cells. The amount of p50-p50 complex decreased after full antigenic stimulation, whereas the amount of the NF-kappa B p50-p65 heterodimer was increased. Increased expression of the IL-2 gene and activity of the IL-2 kappa B DNA binding site correlated with a decrease in the p50-p50 complex. Overexpression of p50 repressed IL-2 promoter expression. The switch from p50-p50 to p50-p65 complexes depended on a protein that caused sequestration of the p50-p50 complex in the nucleus.

Effects of physical exercise on pregnancy outcomes: a meta-analytic review.

In this study, using meta-analysis the findings of all individual reports on the effects of exercise training on pregnancy outcomes were pooled to determine the effects of such training on the pregnant woman and her fetus. Meta-analysis is a statistical technique used to summarize the results of experimental studies that address a common problem. Results show that women who exercised during their pregnancies did not differ from sedentary women for any of the outcome variables measured: maternal weight gain (P = 0.07), infant birth weight (P = 0.20), length of gestation (P = 0.67), length of labor (P = 0.14), and APGAR scores (P = 0.59). Many of the exercise programs exceeded, without apparent adverse effects, the recommended limitations set by the American College of Obstetricians and Gynecologists. Overall, an exercise program using any of a variety of exercise modes that is performed for an average of 43 min.d-1, 3 times.wk-1, at a heart rate of up to 144 bpm, does not appear to be associated with adverse effects to the mother or fetus in a healthy normal pregnancy. However, these findings should be cautiously applied owing to the nature of the currently available data base. Recommendations or precautions for programs of greater intensities can not be made at this time.

Forskolin and prostaglandin E2 regulate the generation of human cytolytic T lymphocytes.

In this paper we examine the characteristics of human cytolytic T lymphocytes (CTL) generated in the presence of forskolin and PGE2. Forskolin and PGE2 suppressed the generation of class-I-specific CTL. The CTL generated in the presence of forskolin and PGE2 had different characteristics which included their ability to proliferate in response to the alloantigen and their lectin-mediated cytolytic activity. The CTL generated in the presence of forskolin had normal proliferative response to the alloantigen, whereas the CTL generated in the presence of PGE2 showed a suppressed proliferative ability to the alloantigen. The two groups of CTL were then tested for their activity in the process of lectin-dependent cell-mediated cytotoxicity. After the addition of PHA into the chromium release assay the CTL generated in the presence of forskolin normally lysed the nonspecific targets, whereas the CTL generated in the presence of PGE2 did not show the normal response in lysing the nonspecific targets. The results suggest that the cytolytic machinery was intact when the CTL were generated in the presence of forskolin but CTL were not able to either recognize or lyse the target cell. However, the CTL generated in the presence of PGE2 did not share the same characteristics as the CTL generated in the presence of forskolin because the CTL generated in the presence of PGE2 were unable to kill even in the presence of lectin. It appears that the inhibitory effects of forskolin were mediated by cAMP and not by its effects on the potassium channels because the 1,9-dideoxy derivative of forskolin which did not activate adenylate cyclase also did not suppress the generation of CTL. However, it was not established whether the diverse effects of PGE2 on the generation of CTL were mediated by cAMP-dependent, -independent or by both mechanisms.