Jane Winer

The Effects of IL-20 Subfamily Cytokines on Reconstituted Human Epidermis Suggest Potential Roles in Cutaneous Innate Defense and Pathogenic Adaptive Immunity in Psoriasis

IL-19, IL-20, IL-22, IL-24, and IL-26 are members of the IL-10 family of cytokines that have been shown to be up-regulated in psoriatic skin. Contrary to IL-10, these cytokines signal using receptor complex R1 subunits that are preferentially expressed on cells of epithelial origin; thus, we henceforth refer to them as the IL-20 subfamily cytokines. In this study, we show that primary human keratinocytes (KCs) express receptors for these cytokines and that IL-19, IL-20, IL-22, and IL-24 induce acanthosis in reconstituted human epidermis (RHE) in a dose-dependent manner. These cytokines also induce expression of the psoriasis-associated protein S100A7 and keratin 16 in RHE and cause persistent activation of Stat3 with nuclear localization. IL-22 had the most pronounced effects on KC proliferation and on the differentiation of KCs in RHE, inducing a decrease in the granular cell layer (hypogranulosis). Furthermore, gene expression analysis performed on cultured RHE treated with these cytokines showed that IL-19, IL-20, IL-22, and IL-24 regulate many of these same genes to variable degrees, inducing a gene expression profile consistent with inflammatory responses, wound healing re-epithelialization, and altered differentiation. Many of these genes have also been found to be up-regulated in psoriatic skin, including several chemokines, β-defensins, S100 family proteins, and kallikreins. These results confirm that IL-20 subfamily cytokines are important regulators of epidermal KC biology with potentially pivotal roles in the immunopathology of psoriasis.

The Vascular Endothelial Growth Factor Proteins: Identification of Biologically Relevant Regions by Neutralizing Monoclonal Antibodies

Angiogenesis plays critical roles in organ development during embryonic and fetal life, wound healing and in a variety of pathological conditions. Vascular endothelial growth factor (VEGF) is a secreted growth factor specific for vascular endothelial cells which induces angiogenesis in vivo. To gain a better understanding of the physiological role of VEGF, we have generated and characterized four murine monoclonal antibodies (mAbs) using the 165 amino acid species of recombinant human VEGF as immunogen. These mAbs (A3.13.1, A4.6.1, B4.3.1 and B2.6.2) belong to IgG1 isotype and have high affinities for VEGF (dissociation constants range from 2.2 x 10(-9) to 4 x 10(-10) M). Two different epitopes were detected with these mAbs. One epitope is recognized by mAbs A3.13.1 and B2.6.2, and the other recognized by mAbs A4.6.1 and B4.3.1. The epitope recognized by mAb A4.6.1 appears to be continuous while mAb B2.6.2 recognizes a discontinuous epitope. MAb A4.6.1 recognized three species of VEGF generated by alternative splicing, VEGF121, VEGF165 and VEGF189 while mAb B2.6.2 binds only VEGF165 and VEGF189. Results using an in vitro bovine adrenal cortex endothelial cell proliferation assay, in in vivo vascular permeability assay and an in vivo embryonic chicken angiogenesis assay showed that mAb A4.6.1 has potent VEGF neutralizing activities. MAb A4.6.1 was shown to block the binding of VEGF to its receptor(s) suggesting the inhibitory mechanism for VEGF activities. These well-defined mAbs should be very powerful tools to understand the structure-function relationship of various domains of VEGF and may have therapeutic potential.

Aortic smooth muscle cells express and secrete vascular endothelial growth factor.

We examined whether cultured bovine aortic smooth muscle (ASM) cells express VEGF. RNA blot analysis of total cellular RNA derived from ASM cells demonstrates the expression of the VEGF gene. ASM cells release in the medium a VEGF-like endothelial cell mitogen which binds to heparin-sepharose and has an apparent molecular weight of 40-45 kDa as assessed by an HPLC gel filtration column. Consistent with VEGF, this mitogen does not stimulate the proliferation of ASM cells. Immunoblot analysis of the bioactive material with an antibody specific for VEGF demonstrates the presence of a major immunoreactive band with an apparent molecular mass of 23 kDa and a minor band with a molecular mass of approximately 18 kDa, in reducing conditions. The major band has very similar apparent molecular weight as the 165 amino-acid species of human recombinant VEGF of folliculo-stellate cells derived VEGF. These data demonstrate the expression and synthesis of VEGF by cultured ASM cells and suggest that the 164 amino-acid species is the predominant molecular form of the growth factor secreted by such cells. VEGF released by ASM cells may play a paracrine role in the maintenance of the integrity of the endothelial lining or in the abnormal proliferation of the vasa vasorum which takes place in atherosclerosis.

Phenylephrine, endothelin, prostaglandin F2α, and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro, and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro

In these studies, we show that endothelin (ET), leukemia inhibitory factor (LIF), phenylephrine (PE), and prostaglandin F2α(PGF2α), which are all hypertrophic for neonatal rat cardiac myocytes in culture, induce distinct morphological, physiological, and genetic changes after a 48-h treatment. Transmission electron microscopy revealed differences in myofibril organization, with ET-treated cells containing the most mature-looking myofibrils and PGF2α — and LIF-treated cells the least. ET- and PE-treated cultures contained the same number of beating cells as control, but LIF and PGF2α treatment increased the number of beating cells 180%. Treatment with LIF, PE, and PGF2α increased the beat rate to 3.3 times that of control. After exposure to the β-adrenergic agonist isoproterenol, the beat rate increased 50% for PGF2α, 54% for PE, 84% for LIF, and 125% for control. ET treatment did not increase the beat rate, nor did these cells respond to isoproterenol. ET, LIF, and PE increased the production of atrial natriuretic peptide (ANP) by three-fold and PGF2α by 18-fold over nontreated cells. Brain natriuretic peptide (BNP) was increased fourfold by ET and PE, 16-fold by LIF, and 29-fold by PGF2α. Interestingly, on a pmol/L basis, only LIF induced more BNP than ANP. Treatment with all agents led to a similar pattern of gene induction: increased expression of the embryonic genes for ANP and skeletal α-actin, and less than a twofold change in the constitutively expressed gene myosin light chain-2, with the exception that LIF did not induce skeletal α-actin. Each agent, however, induced ANP mRNA with a different time-course. We conclude that at least four distinct cardiac myocyte hypertrophy response programs can be induced in vitro. Further studies are necessary to determine whether these correlate to the different types of cardiac hypertrophy seen in vivo.

Cardiac fibroblasts produce leukemia inhibitory factor and endothelin, which combine to induce cardiac myocyte hypertrophy in vitro.

Cardiac fibroblasts in culture produce factor(s) that induce hypertrophy of neonatal rat ventricular myocytes in vitro. As in vivo, the myocyte hypertrophy response in culture is characterized by an increase in cell size and contractile protein content, and by the activation of embryonic genes, including the gene for atrial natriuretic peptide. The purpose of this study was to identify the factor(s) produced by fibroblasts that induce myocyte hypertrophy. The fibroblast hypertrophy activity was inhibited using a combination of the endothelin A receptor blocker BQ-123 and an antibody to leukemia inhibitory factor. The individual antagonists each caused a partial inhibition. The mRNAs for both leukemia inhibitory factor and endothelin were detected by RT-PCR analysis and the concentration of both proteins was determined to be approximately 200 pmol/L in the conditioned medium using immunoassays. Purified leukemia inhibitory factor and endothelin each induced distinctive morphological changes in the myocytes. Their combination generated a different morphology similar to that induced by fibroblast conditioned medium. Each factor also induced atrial natriuretic peptide production, but both were required for the myocytes to produce the levels measured after exposure to fibroblast conditioned medium. These results show that hypertrophy activity produced by cardiac fibroblasts in culture is a result of leukemia inhibitory factor and endothelin.

Development and Application of Real-Time Quantitative PCR

Since the original description of the polymerase chain reaction (PCR), novel applications of this technique have been reported at an exponential rate. The power of the method lies in the ability to analyze minute amounts of samples (e. g., dried blood, microliters of liquid, several cells) or even to detect a single molecule. Virtually every area of biological science has been impacted by PCR, including forensics, genetics, clinical diagnostics, drug discovery, and environmental biology.

Endogenous cardiac vasoactive factors modulate endothelin production by cardiac fibroblasts in culture.

Endothelin, a potent vasoconstrictor, is produced by cardiac fibroblasts in culture and induces hypertrophy in cardiac myoctes. The purpose of this study was to determine whether vasoactive factors endogenous to the heart affect the production of endothelin by cultured cardiac fibroblasts. Vasoactive factors have been shown to play multiple roles in the adaptation of the heart to chronic overload, affecting both vascular tone and cell growth. Both atrial (ANP) and brain (BNP) natriuretic peptides are endogenous cardiac vasodilators and are produced by cultured myocytes in response to stimulation with endothelin. Treatment of cardiac fibroblasts with these peptides decreased endothelin production. Nitroprusside, an activator of guanylyl cyclase, decreased endothelin production indicating the involvement of cGMP in the response. Carbaprostacyclin, a stable derivative of prostacyclin, another endogenous cardiac vasodilator, also decreased endothelin production by fibroblasts. The combination of BNP and carbaprostacyclin was additive in decreasing endothelin production. In contrast, PGF2α and angiotensin II, both endogenous cardiac vasoconstrictors, increased endothelin production and overcame the inhibition induced by BNP and carba-prostacyclin. In summary, endothelin production by cardiac fibroblasts was decreased by the endogenous cardiac vasodilators ANP, BNP, and prostacyclin and increased by the endogenous vasoconstrictors PGF2α and angiotensin II.

Herceptin-sensitivity of HER2-transgenic mouse mammary tumors

The long-term goal of our work is to better understand the mechanisms of Herceptin action and resistance. Toward that end, we have produced transgenic mice that express HER2 under the control of the mouse mammary tumor virus (MMTV) promoter. Transgenic mouse models offer the advantage of having immune system components that may be important in the action of antibodies but are lacking in immunodeficient hosts. ErbB2 transgenic mice have been produced by Muller and colleagues using the rat homolog neu, which does not bind Herceptin. Therefore, we made mice using the human version, HER2. These mice express HER2 in the mammary gland at very high levels and develop HER2-positive mammary tumors within 6–8 months. Several tumors have been propagated as allografts in wild-type mice by implanting a small piece of tumor into the mammary fat pad. Cohorts of mice bearing the same tumor line were then treated with 4D5, the murine version of Herceptin, or vehicle. Using this approach, we found that several tumor lines are completely Herceptin resistant, others are growth inhibited, and one regresses completely. As observed previously in tumors from neu transgenic mice, all of our HER2 tumors contain small deletions in the juxtamembrane region of the HER2 transcript. Within a tumor line, each mutation is unique and remains stable on continued passage. It is unclear, however, whether such mutations are responsible for determining Herceptin sensitivity. Gene expression profiling between the highly sensitive tumor line (F2-1282) and a Herceptin-resistant line (Fo5) revealed a large number of differences that could contribute to resistance. Gene expression profiling is also being used to identify changes that take place in F2-1282, the Herceptin-sensitive tumor, when it is exposed to Herceptin/4D5. In summary, like individual breast cancer patients, tumors from MMTV-HER2 mice display the entire spectrum of responsiveness to Herceptin/4D5, from resistant to complete regression, and therefore may help provide insights into cofactors that are important for Herceptin activity.

Chapter 2.2.4 Application of real-time RT-PCR for quantification of gene expression

Publisher Summary This chapter discusses the application of real-time RT-PCR for quantification of gene expression. The use of real-time RT-PCR methodology permits rapid assay development from validation of RNA sample preparation methods, to validation of the invariance of housekeeping genes. Real-time PCR also provides a quantitative method for moderate to high throughput analysis of gene expression. The ABI instrument permits the analysis of 96 tubes simultaneously, allowing for multiple samples and genes to study in single PCR experiment. The ABI instrument permits the analysis of 96 tubes simultaneously, allowing for multiple samples and genes to be studied in single PCR experiment. This system can produce precise and reliable data which correlates with traditional methods. In addition, the chapter illustrates that real-time RT-PCR technology plays a major role in the future of quantitative gene expression analysis.

Chapter 3.2.7 Protein targeting in the functional analysis of EphA receptors: the use of immunoadhesins

Publisher Summary This chapter describes application of immunoadhesins, perhaps most relevant for the behavioral neuroscientist. It focuses on a particular protein family (EphA receptor tyrosine kinases) and the investigation of their potential role in certain aspects of behavior and brain function. The chapter is intended to be an example to delineate the advantages along with the disadvantages of using immunoadhesins, a method novel in behavioral neuroscience. In addition, the present findings revealed a role for EphA receptor tyrosine kinases in cognitive function in the adult mammalian brain. These findings open an unexpected avenue into the functional analysis of this large receptor protein family and may lead to novel targets for therapeutic intervention in human brain and behavioral disorders. They also demonstrate that protein targeting with the application of immunoadhesins may be a useful research strategy in the analysis of molecular components involved in such complex behavioral traits as learning and memory.