Lawrence B. Lachman

Real-Time Quantitative PCR for Detection of Helicobacter pylori

ABSTRACT Helicobacter pylori is one of the most common chronic infections in humans, in whom it is a key etiological factor in peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Humans are the bacteriums only host. Here we report the development of a real-time quantitative (Q) PCR-based assay to measure ureC gene copy number to detect H. pylori, based on the fact that there is only one copy of the ureC gene per bacterium. Upon optimization of LightCycler Q-PCR conditions, we obtained a standard curve with a linear range (correlation coefficient = 1) across six logs of DNA concentration. We were able to accurately quantify as few as 1,000 bacteria in our assay. Analysis of variance on 15 randomly selected clinical samples showed good reproducibility of this assay. Comparison of Q-PCR results with bacterial culture and histopathological results from an additional 85 clinical biopsy samples showed a significant difference for the presence of H. pylori. Many samples that were negative for H. pylori by culture and histopathology were positive by Q-PCR. Contamination of PCR by H. pylori or H. pylori genetic material could not be ruled out. In summary, we developed a rapid, sensitive, and real-time Q-PCR method for detecting H. pylori. This technique offers a significant improvement over other available methods for detecting H. pylori in clinical and research samples.

DNA vaccination against neu reduces breast cancer incidence and metastasis in mice.

The gene for HER2/neu is overexpressed in 30–40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. The HER2/neu gene product, a transmembrane protein kinase member of the EGF receptor family, has significant potential as a tumor antigen for vaccination. We inserted the sequence for neu into a novel plasmid called ELVIS containing a Sindbis virus replicon that reproduces multiple copies of mRNA. Mice vaccinated one time intramuscularly demonstrated a strong antibody response against A2L2, a murine breast cancer cell line transfected to express neu. Vaccinated mice challenged in the mammary fatpad with A2L2 had reduced tumor incidence and reduced tumor mass compared to mice challenged with tumor cells lacking the neu insert. Intradermal vaccination was also protective and required 80% less plasmid for a similar level of protection. Vaccination reduced the incidence of lung metastasis from mammary fatpad tumors and reduced the number of lung metastases resulting from intravenous injection of A2L2 cells. Cytotoxic T lymphocytes cultures of immune spleen cells with P815-neu cells produced high levels of interferon-γ indicating an antigen-specific Th1-type immune response resulting from the vaccination. In a spontaneous breast tumor model using neu transgenic mice, vaccination with ELVIS-neu protected against development of spontaneous breast tumors. Our preclinical data indicate that therapeutic vaccination of patients with ELVIS-neu may reduce metastasis from HER2/neu-expressing breast and ovarian tumors. Cancer Gene Therapy (2001) 8, 259–268

[36] Interleukin 1 from human leukemic monocytes

Publisher Summary Interleukin 1 (IL-1) is a pluripotent, hormone-like peptide that affects many different cell types and organ systems. IL-1 is primarily produced by monocytes and macrophages, although other cell types release IL-1- like factors. The purification of IL-1 is hampered by the difficulties associated with obtaining large quantities of human monocytes. Residual leukocytes that result from various blood banking procedures are the major source of human monocytes. An alternative source of human monocytes is leukemia cells from patients with AMoL or AMML. The chapter further discusses murine thymocyte assay for IL-1. The routine thymocyte assay for IL-1 is a sensitive, reproducible assay for the measurement of IL-1. Murine thymocytes were cultured in the presence either of PHA or Con A for maximum response to IL-1.7 Subsequent experimentation demonstrated that murine thymocytes of precise age would, in fact, respond mitogenically to IL-1 in the absence of lectin. Although several strains and ages of mice have been investigated, one mouse strain has produced consistent results since 1978. The chapter further explains preparation of IL-1-containing conditioned medium.

Elastography imaging of small animal oncology models: a feasibility study

To test the feasibility of applying ultrasonic elastography on small animal oncology models, experiments were performed in vitro and in situ on murine mammary lesions induced exogenously by tumor cell line 66.3. In vitro studies involved three 1-week-old excised tumors embedded in a phantom block with ultrasonic properties similar to those of soft biologic tissues. In situ studies involved five mice whose bodies were embedded in pure gelatin blocks. The data were acquired from the blocks with a clinical scanner modified to have an automated compressor assembly and processed to construct the elastograms at various imaging planes within each block. The results were analyzed both qualitatively and quantitatively to assess the merits of the elastographic imaging and its limitations for in vivo serial studies of tumors in small animal oncology models.

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1α) and tumor necrosis factor alpha (TNFα) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1α and TNFα were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNFα-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1α and TNFα significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1α and TNFα displayed significant in vivo antitumor activity against the IL1α- and TNFα-resistant B16F10 metastatic murine melanoma.

Human Immunoregulatory Molecules: Interleukin 1, Interleukin 2. and B-Cell Growth Factor

Intensive investigations over the past several years have clearly indicated that a significant degree of immune regulation is accomplished by soluble mediators. Among the host of potential immune regulators that have been described to date, the interleukins have been the most fully characterized. Yet many aspects of both the biochemistry and the cell biology of these factors remain to be determined. In an attempt to place some of the areas of current investigational interest into perspective, a brief historical overview of each factor will be undertaken. This historical overview will detail our knowledge of the biochemical properties attributed to each factor when first described. This latter point will be of significance in our subsequent attempts to unravel the biological properties of each factor and how it mediates those effects that lead to a competent immune response.

Development of a Murine Model of Helicobacter pylori Infection

A murine model for Helicobacter pylori infection could facilitate vaccine development. This study was designed to determine the effect of various conditions of dose, frequency of administration, and fasting on H. pylori infection of mice.

Induction of IL-1: Independent production of IL-1α and IL-1β

Abstract Lipopolysaccharide (LPS) either in its soluble form or associated with multilamellar phospholipid vesicles (liposomes) was investigated for its ability to induce human monocyte interleukin (IL)-1α and IL-1β. When human monocytes were activated in vitro by LPS either in its soluble form or presented at the surface of lyophilized multilamellar vesicles (Lyo-MLV-LPS), both IL-1α and IL-1β were detected intracellularly and extracellularly, using specific antisera. In correlation with these findings, the mRNAs for IL-1α and IL-1β were both found by Northern blot analysis. However, when human monocytes were stimulated by LPS incorporated into multilamellar vesicles which had not been previously lyophilized, a different pattern of IL-1 protein and message was observed. IL-1α activity was detected only intracellularly and not in the supernatant, while IL-1β was not produced at all. Northern blotting revealed only mRNA for IL-1α as soon as 0.5 h after stimulation and none for IL-1β. These data indicate independent induction of IL-1α and IL-1β. Moreover, it appears that the regulation occurs at the transcriptional level, since with MLV-LPS only the mRNA for IL-1α was induced. The lack of IL-1β could be due to either a blockage at the DNA level, an undetectable level of IL-1β mRNA, or a very short halflife for IL-1β mRNA. These findings indicate that although IL-1α and IL-1β may have identical biological properties and share the same receptor, their induction and secretion are regulated by independent pathways. Moreover, the same activator, presented in a different manner to the same cells can induce different consequences.

Liposomal cytokines and liposomes targeted to costimulatory molecules as adjuvants for human immunodeficiency virus subunit vaccines

Publisher Summary The use of adjuvants is critical for the development of successful subunit vaccines because most peptide antigens are either non-immunogenic or weakly immunogenic. Adjuvants provide a mechanism for antigen persistence at the injection site and enhance the immune response to immunogens by prolonging their release and time of interaction with antigen presenting cells (APC). Cytokines have significant potential as adjuvants, because most adjuvants function by inducing the production of cytokines from cells of the immune system. Cytokines—such as interleukin (IL)-1, IL-2, IL-6, IL-12, and IFN-γ—have been used as adjuvants. Liposomes can be used for their depot effect to release antigens and other incorporated adjuvants, to increase antigen uptake by targeting antigens, and to generate strong immune responses. The identification of B7-1 (CD80) and B7-2 (CD86) as the prototypic costimulatory molecules has led to new strategies for immunotherapy and vaccination. T-cell activation requires signals from the antigen-specific T-cell receptor (TCR) and the antigen-independent CD28 receptor. T-cell activation leads to the secretion of proinflammatory cytokines and effector T-cell functions. The manipulation of the TCR-costimulatory receptor network offers therapeutic opportunities for the control of hyper-responsiveness, such as autoimmune diseases and for the control of hyporesponsiveness as exhibited by a poor response to subunit vaccine antigens and tumor antigens.

Summary of the Interleukin 1 Workshop Discussions

The great number of abstracts which were received for the Interleukin 1 (IL 1) Workshop necessitated, that the session be held in two parts on separate days. Each individual session consisted of individual presentations and a panel discussion.