Timothy K. Hart

Immunomodulatory Effects of Anti-CD4 Antibody in Host Resistance against Infections and Tumors in Human CD4 Transgenic Mice

ABSTRACT Anti-CD4 antibodies, which cause CD4+ T-cell depletion, have been shown to increase susceptibility to infections in mice. Thus, development of anti-CD4 antibodies for clinical use raises potential concerns about suppression of host defense mechanisms against pathogens and tumors. The anti-human CD4 antibody keliximab, which binds only human and chimpanzee CD4, has been evaluated in host defense models using murine CD4 knockout-human CD4 transgenic (HuCD4/Tg) mice. In these mice, depletion of CD4+ T cells by keliximab was associated with inhibition of anti-Pneumocystis carinii and anti-Candida albicans antibody responses and rendered HuCD4/Tg mice susceptible to P. carinii, a CD4-dependent pathogen, but did not compromise host defense against C. albicans infection. Treatment of HuCD4/Tg mice with corticosteroids impaired host immune responses and decreased survival for both infections. Resistance to experimental B16 melanoma metastases was not affected by treatment with keliximab, in contrast to an increase in tumor colonization caused by anti-T cell Thy1.2 and anti-asialo GM-1 antibodies. These data suggest an immunomodulatory rather than an overt immunosuppressive activity of keliximab. This was further demonstrated by the differential effect of keliximab on type 1 and type 2 cytokine expression in splenocytes stimulated ex vivo. Keliximab caused an initial up-regulation of interleukin-2 (IL-2) and gamma interferon, followed by transient down-regulation of IL-4 and IL-10. Taken together, the effects of keliximab in HuCD4/Tg mice suggest that in addition to depleting circulating CD4+ T lymphocytes, keliximab has the capability of modulating the function of the remaining cells without causing general immunosuppression. Therefore, keliximab therapy may be beneficial in controlling certain autoimmune diseases.

A Novel Technique For Mapping the Lipid Composition of Atherosclerotic Fatty Streaks by En Face Fluorescence Microscopy

We introduce here a new fluorescence microscopy technique for en face analysis of the atherosclerotic fatty streaks (FS). This technique is semiquantitative and has the sensitivity and resolution to map lipids to individual cells in FS less than 100 μm in diameter. New Zealand White rabbits were fed an atherogenic diet for up to 26 weeks. Aortas were fixed in formalin and stained en bloc with the fluorescent dyes Nile red and filipin. Fluorescent staining was validated by correlating microfluorimetric and biochemical measurements of the lipid content in FS. To determine the cell types associated with the different staining patterns, FS were also evaluated by transmission electron microscopy (TEM) and immunohistochemistry (IH). Correlation of microfluorimetry, TEM, IH, and biochemical data indicated that regions rich in non-esterified cholesterol stained with filipin and fluoresced blue owing to accumulations of lipid vessicles and/or cholesterol crystals. Regions rich in neutral and polar lipids stained with Nile red and fluoresced yellow or orange, respectively, owing to accumulations of lipids in both macrophages and smooth muscle cells (SMC). Digital overlays of the filipin and Nile red images revealed that larger lesions (>0.5 mm diameter) had a “nested” distribution of lipids, with a blue (filipin) fringe surrounding an orange (Nile red) fringe surrounding a yellow (Nile red) center.

Preclinical Safety of Recombinant Human Interleukin-18

Recombinant human interleukin-18 (rHuIL-18) is currently in clinical trials for treatment of cancer. This report presents results of preclinical toxicity studies with rHuIL-18 in cynomolgus monkeys and recombinant murine IL-18 (rMuIL-18) in mice. The rHuIL-18 was administered intravenously in 1 or 2 different 5-day cycles at doses 0.3 to 75 mg/kg/day in monkeys. Decreases in red cell mass, neutrophil, and platelet counts, increases in monocyte and large unstained cell counts, and lymphoid hyperplasia in spleen and lymph nodes were mild, reversible, and likely related to the pharmacologic activity of IL-18. The only toxic effect was protein cast nephropathy, secondary to coprecipitation of administered IL-18 and Tamm-Horsfall protein in the distal nephron, that only occurred at 75 mg/kg/day. Other adverse effects of rHuIL-18 were related to strong immunogenicity in monkeys and were manifest only during a second dosing cycle. The rMuIL-18, at similar dosing levels and cycles in mice, resulted in reduced red cell mass, increased white blood cell counts, spleen and lymph node hyperplasia, and mild, reversible changes in intestine, liver, and lungs. Protein cast nephropathy occurred in mice at doses ≥30 mg/kg/day. In conclusion, preclinical safety studies showed that rIL-18 was well tolerated at pharmacologically active doses in both monkeys and mice.

Morphometric analysis of envelope glycoprotein gp120 distribution on HIV-1 virions.

The surface of HIV-1, like that of other retroviruses, is studied with virally encoded glycoproteins which appear ultrastructurally as electron-dense spikes or knobs. The glycoprotein that forms the spike structure, gp120, is non-covalently bound to the transmembrane glycoprotein gp41. Mature HIV-1 virions do not have as many spikes as the genetically related retroviruses HIV-2 and SIV. gp120 is lost from HIV-1 during viral morphogenesis and after incubation of the virus with the soluble form of cellular receptor CD4. In this study we used ultrastructural cytochemistry and morphometry to quantitate the distribution of envelope glycoprotein spikes on budding and mature HIV-1 virions and to look for alternatives to the laborious and somewhat subjective spike-counting technique for envelope spike analysis on HIV-1. HIV-1, strain HTLV-IIIB, was examined after staining of envelope glycoproteins with either tannic acid, immunogold staining for gp120 (gp120-immunogold), or lectin-gold staining with concanavalin A for mannose residues (ConA-HRP-gold) and frequency distributions of spikes or gold particles per micron HIV-1 membrane generated. Envelope spikes were normally distributed on membranes of budding and mature HIV-1. However, the density of spikes per micron viral membrane on mature HIV-1 virions was approximately 50% of that observed on budding virions. ConA-HRP-gold and gp120-immunogold did not efficiently label budding virions. The shape of the frequency distribution for ConA-HRP-gold particles on mature virions was similar to that for envelope spikes and could be used to quantitate envelope glycoproteins on HIV-1. In addition, ConA-HRP-gold staining was able to detect the loss of envelope proteins after treatment of virus with soluble CD4. gp120-immunogold labeling was patchy and many virions were unlabeled. ConA-HRP-gold staining proved to be a rapid, reliable, and easily quantifiable method for estimation of envelope glycoprotein density on mature HIV-1. However, the loss of spike structures throughout the life cycle of HIV-1 can effectively be determined only by direct spike counting.

Flow cytometry in the preclinical development of biopharmaceuticals.

Novel biomarkers are often required in the preclinical development of biopharmaceuticals in order to characterize pharmacologic and toxicologic effects and to establish pharmacodynamic and pharmacokinetic relationships. Flow cytometry is uniquely suited for measurement of these biomarkers. Large numbers of single cells in a heterogeneous population can be rapidly identified and characterized with high accuracy and reproducibility. Cells are not damaged by the detection system and can be subsequently sorted for further morphologic or functional analysis. The availability of clinical instruments and a wide range of fluorescent probes have made this technology applicable for use in toxicologic clinical pathology. Flow cytometry has played an integral role in the development of a monoclonal antibody to human CD4 (keliximab, IDEC-CE9.1, SB 210396). Lymphocyte subset analysis and assays for expression, coating, and modulation of human CD4 were used for sequential assessment of the pharmacologic activity of keliximab in transgenic mice expressing human CD4.

Quantitative Immunocytochemical Staining for Recombinant Tissue-Type Plasminogen Activator in Transfected Chinese Hamster Ovary Cells

Abstract Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA In CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.