Clinical applications of interleukin-2

Interleukin-2 (IL-2) is a cytokine with potent immunomodulating properties which has shown considerable antitumour activity in preclinical models. In clinical trials, the effects of IL-2 given by various routes and schedules have been investigated. IL-2 has been administered either as single drug or in combination with other cytokines and immunomodulating agents, chemo therapeutic agents, or reinfusions of ex vivo activated autologous cytotoxic effector cells. The results of published clinical studies with IL-2 based immunotherapy are reviewed in this paper.     

Mechanisms,function and clinical applications of DNp73

p73, has two distinct promoters, which allow the formation of two protein isoforms: full-length transactivating (TA) p73 and an N-terminally truncated p73 species (referred to as DNp73) that lacks the N-terminal transactivating domain. Although the exact cellular function of DNp73 is unclear, the high expression levels of the genes have been observed in a variety of human cancers and cancer cell lines and have been connected to pro-tumor activities. Hence the aim of this review is to summarize DNp73 expression status in cancer in the current literature. Furthermore, we also focused on recent findings of DNp73 related to the biological functions from apoptosis, chemosensitivity, radiosensitibity, differentiation, development, etc. Thus this review highlights the significance of DNp73 as a marker for disease severity in patients and as target for cancer therapy.     

Functions, applications and production of 2-O-d-glucopyranosyl-l-ascorbic acid

Vitamin C (VC) is an essential nutrient that cannot be synthesized by the human body. Due to its extreme instability, various VC derivatives have been developed in an attempt to improve stability while retaining the same biological activity. One of the most important VC derivatives, 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G), has attracted increasing attention in recent years with a wide range of applications in cosmetics, food, and medicine. In this mini-review, we first introduce the types and properties of different VC glycosyl derivatives. Next, we provide an overview of the functions and applications of AA-2G. Finally, we discuss in-depth the current status and future prospects of AA-2G production by biotransformation.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

Regulation of lymphocyte proliferation by uterine serpin: interleukin-2 mRNA production, CD25 expression and responsiveness to interleukin-2

During pregnancy, the endometrium of the ewe secretes large amounts of a progesterone-induced protein of the serpin superfamily of serine proteinase inhibitors called ovine uterine serpin (OvUS). This protein inhibits lymphocyte proliferation in response to concanavalin A (ConA), phytohemagglutinin (PHA), or mixed lymphocyte reaction. The purpose of these experiments was to characterize the mechanism by which OvUS inhibits lymphocyte proliferation. Ovine US caused dose-dependent inhibition of lymphocyte proliferation induced by phorbol myristol acetate (PMA), an activator of protein kinase C. The PHA-induced increase in CD25 expression was inhibited in peripheral blood mononuclear leukocytes (PBML) by OvUS. However, no effect of OvUS on Con A-induced expression of CD25 was observed. Further analysis using two-color flow cytometry revealed that OvUS inhibited ConA-induced expression of CD25 in gammadelta-TCR- cells but not gammadelta-TCR+ cells. Stimulation of PBML for 14 hr with ConA resulted in an increase in steady state amounts of interleukin-2 (IL-2) mRNA that was not inhibited by OvUS. Ovine US was also inhibitory to lymphocyte proliferation induced by human IL-2. Results suggest that OvUS acts to inhibit lymphocyte proliferation by blocking the upregulation of the IL-2 receptor and inhibiting IL-2-mediated events. Lack of an effect of OvUS on ConA-stimulated CD25 expression in gammadelta-TCR+ cells may reflect a different mechanism of activation of these cells or insensitivity to inhibition by OvUS.     

Animal-free production of ccc-supercoiled plasmids for research and clinical applications

The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)-an important tool for quality control and stability assessments in DNA storage or application. Hence, a large-scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal-derived substances. Using CGE, the development and in-line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP). Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection). The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms.     

Stem cell bioprocess engineering towards cGMP production and clinical applications

Stem cells, including mesenchymal stem cells and pluripotent stem cells, are becoming an indispensable tool for various biomedical applications including drug discovery, disease modeling, and tissue engineering. Bioprocess engineering, targeting large scale production, provides a platform to generate a controlled microenvironment that could potentially recreate the stem cell niche to promote stem cell proliferation or lineage-specific differentiation. This survey aims at defining the characteristics of stem cell populations currently in use and the present-day limits in their applications for therapeutic purposes. Furthermore, a bioprocess engineering strategy based on bioreactors and 3-D cultures is discussed in order to achieve the improved stem cell yield, function, and safety required for production under current good manufacturing practices.     

Polyamine biosynthesis is necessary for interleukin-2-dependent proliferation but not for interleukin-2 production or high-affinity interleukin-2 receptor expression

DL-alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), inhibited concanavalin A-induced proliferation of splenic mononuclear cells (SMNC). The inhibition was not reversed by interleukin-2 (IL-2) addition. Although DFMO did not affect the production of IL-2 or the expression of high-affinity IL-2 receptor, IL-2-dependent proliferation of SMNC was inhibited by DFMO, and the inhibition was reversed by exogenous putrescine. The inhibition of IL-2-dependent DNA synthesis appeared to be related to the decrease in intracellular polyamines. When the proliferation of SMNC was induced by IL-2, ODC activity was also increased. A similar result was obtained in the proliferation of an IL-2-dependent T cell line, CTLL. The time course of ODC induction was similar to that of IL-2 production by concanavalin A-stimulated SMNC. These results indicate that polyamine biosynthesis is necessary for IL-2-dependent proliferation, but not for IL-2 production or IL-2 receptor expression.     

Regulation of interleukin-2 and interleukin-6 production from T-cells: involvement of interleukin-1 beta and transforming growth factor-beta

The effect of recombinant (r) interleukin-1 beta (rIL-1 beta) and transforming growth factor-beta (TGF-beta) on the production of interleukin-2 (IL-2) and interleukin-6 (IL-6) from an antigen-specific (LBRM-33-1A5) and an antigen-nonspecific (EL-4-NOB-1) T-cell line was investigated. rIL-1 beta induced the production of IL-2 and IL-6 from EL-4-NOB-1 cells in a dose-related manner. The LBRM-33-1A5 cells required phytohemagglutinin (PHA) in addition to rIL-1 beta in order to produce IL-2 and IL-6. IL-2 production was found to precede IL-6 production in both cell lines. No IL-2 or IL-6 production was observed by adding r murine tumor necrosis factor-alpha or r murine interferon gamma to the cells. The presence of 1 ng/ml TGF-beta reduced IL-2 and IL-6 production from both T-cell lines by more than 80%. The inhibition of IL-2 and IL-6 production was still evident by a concentration as low as 10 pg/ml of TGF-beta. rIL-1 beta and PHA also stimulated murine thymocytes to produce IL-6 which was inhibited up to 85% in the presence of 1 ng/ml TGF-beta. Taken together these results suggest that TGF-beta may suppress immune responses by inhibiting the endogenous production of IL-2 and IL-6.     

Pullulan: biosynthesis,production, and applications

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium pullulans, which has long been applied for various applications from food additives to environmental remediation agents. This review article presents an overview of pullulan’s chemistry, biosynthesis, applications, state-of-the-art advances in the enhancement of pullulan production through the investigations of enzyme regulations, molecular properties, cultivation parameters, and bioreactor design. The enzyme regulations are intended to illustrate the influences of metabolic pathway on pullulan production and its structural composition. Molecular properties, such as molecular weight distribution and pure pullulan content, of pullulan are crucial for pullulan applications and vary with different fermentation parameters. Studies on the effects of environmental parameters and new bioreactor design for enhancing pullulan production are getting attention. Finally, the potential applications of pullulan through chemical modification as a novel biologically active derivative are also discussed.     

Concurrent production of interleukin-2, interleukin-10, and gamma interferon in the regional lymph nodes of mice with influenza pneumonia.

Cytokine production has been assessed at the single-cell level (ELISPOT assay) for freshly isolated mediastinal lymph node cells from C57BL/6 mice with primary, nonfatal influenza pneumonia. The mediastinal lymph node populations were also secondarily stimulated in vitro, and culture supernatants were assayed by enzyme-linked immunosorbent assay. Both approaches showed minimal evidence of protein secretion for interleukin-4 (IL-4), IL-5, and tumor necrosis factor, while IL-2, IL-10, and gamma interferon (IFN-gamma) were prominent throughout the response. The numbers of IL-2- and IFN-gamma-producing cells were maximal at 7 days after infection, while the total counts for cells secreting IL-10 were fairly constant from day 3 to 7. The cultures that were stimulated with virus in vitro showed in inverse relationship between IL-10 and IFN-gamma production, with IL-10 peaking on day 3 and IFN-gamma peaking on day 7. Lymphocytes secreting IL-2, IL-10, and/or IFN-gamma were present in CD4+ and CD8+ populations separated by fluorescence-activated cell sorting, although the CD8+ T cells produced less cytokine and were at a relatively lower frequency. Addition of recombinant IL-10 to the virus-stimulated cultures decreased the amount of IFN-gamma that could be detected, while incorporation of a monoclonal antibody to IL-10 had the opposite effect. A neutralization experiment also indicated that IL-2 was the principal mediator of lymphocyte proliferation. These experiments thus show that the developing T-cell response in the regional lymph nodes of mice with influenza cannot be rigidly categorized on the basis of a TH1 or TH2 phenotype and suggest possible regulatory mechanisms.     

Genetic control of interleukin-2 production in inbred mice

BALB/c mice (H-2d haplotype) produced IL 2 better than C57BL/6 mice (H-2d haplotype). However genetic analysis in F2 generation demonstrated independent segregation of the IL 2 production intensity and H-2 haplotype. Investigation of the IL 2 production intensity in BALB/c, C57BL/6 and their F1 and F2 generation revealed that this was controlled by only one gene.     

Lessons from the clinical trials of interleukin-2

The initial results of interleukin-2 (IL-2) therapy in man are reviewed from the perspective of how they conform to predictions from preclinical studies. These preclinical models predict that tumors will vary in their susceptibility to IL-2 therapy and will be most successfully treated at lower tumor burdens. In addition, the dose and schedule of IL-2 are important for successful therapy. Host-related factors, including the presence of suppressor activities, may also be important. In these models, the addition of other cytokines, including interferon-alpha or tumor necrosis factor, to IL-2 can enhance antitumor activity. The concomitant administration of ex vivo IL-2-activated lymphokine-activated killer cells or tumor-infiltrating lymphocytes also enhances the IL-2 antitumor effect. Clinical trials addressing all of these issues have been completed or are underway; the results suggest overall that the preclinical models are predictive, with both host- and tumor-related factors as well as such IL-2-therapy-related factors as dose, schedule, route and the use of additional agents all playing a role in the success of therapy. A more complete understanding of the mechanisms of response and resistance involved in this therapy will facilitate the rational development of more effective and less toxic IL-2-based therapy of human malignancy.     

Expression and production of recombinant human interleukin-2 in potato plants

Interleukin-2 is a pharmacologically important cytokine secreted by T lymphocytes. Recombinant interleukin-2 has been produced and found to be useful for many medical applications. Mass production of recombinant interleukin-2 will be prerequisite for a wider application of this molecule. In this study we investigated the possibility of using potato tubers for the production of recombinant human interleukin-2 in large quantity. A binary vector carrying the human interleukin-2 gene under a potato tuber-specific promoter (patatin promoter) was constructed. Several potato transformants expressing the human interleukin-2 gene were generated by Agrobacterium-mediated transformation. Expression of the human interleukin-2 gene was confirmed by Northern blotting and the protein level was determined by Western blot analyses. A bioassay revealed that human interleukin-2 expressed in the potato tuber supported proliferation of interleukin-2-dependent cells, CTLL-2. We found that the recombinant protein in the 2-week-old microtuber has the highest activity (115 units per gram of microtuber) and estimated that an average yield for a potato (average 200 g per potato) was 23,000 units of rhIL-2 activity. The results suggest that the potato tuber is an excellent system for the mass production of biologically active human interleukin-2.     

The production of two Th2 cytokines, interleukin-4 and interleukin-10, is controlled independently by locus Cypr1 and by loci Cypr2 and Cypr3, respectively

 The strains BALB/cHeA (BALB/c) and STS/A (STS) differ in production of IL-4 and IL-10, two Th2 cytokines, after stimulation of spleen cells with Concanavalin A, STS being a low and BALB/c a high producer. We analyzed the genetic basis of this strain difference using the recombinant congenic (RC) strains of the BALB/c-c-STS/Dem (CcS/Dem) series. This series comprises 20 homozygous strains. Each CcS/Dem strain contains a different, random set of approximately 12.5% genes of the "donor" strain STS and approximately 87.5% of the "background" strain BALB/c. We selected for further analysis the RC strain production intermediate between BALB/c and STS. In (CcS-20×BALB/c)F₂ hybrids we found that different loci control expression of IL-4 and IL-10. Cypr1 (cytokine production 1) on chromosome 16 near D16Mit15 controls IL-4 production, whereas the production of IL-10 is influenced by loci Cypr2 near D1Mit14 and D1Mit227 on chromosome 1 and Cypr3 marked by D5Mit20 on chromosome 5. In addition, the relationship between the level of these two cytokines depends on the genotype of the F₂ hybrids at a locus cora1 (correlation 1) on chromosome 5. This differential genetic regulation may be relevant for the understanding of biological effects of T-helper cells in mice of different genotypes. Received: 2 March 1998 / Revised: 8 June 1998