Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model

Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis^®), a humanized IgG₁ monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2–5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p < 0.001 for both mAbs) and therapeutic protocols (p < 0.001 and p < 0.05 respectively). The additional plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.     

Loss of organic acids, amino acids, k, and cl from barley roots treated anaerobically and with metabolic inhibitors

Excised roots of barley (Hordeum vulgare, var. Campana) lost organic acids, amino acids, K⁺, and Cl⁻ within 15 minutes after initiation of anaerobic treatment or treatment with NaCN and 2,4-dinitrophenol. Initial loss of organic acids when roots were placed under N₂ is attributed to a decarboxylation reaction, possibly catalyzed by phosphoenolpyruvate carboxykinase. Organic and amino acids began to leak from the roots to the bathing medium after 1 to 2 hours under N₂, indicating injury to cell membranes. During the first hour of anaerobic treatment, K⁺ loss from low-salt roots was equivalent to organic acid loss. Potassium loss from roots containing high levels of KCl was approximately equal to organic acid plus amino acid loss; and Cl⁻ loss was approximately equal to amino acid loss. It is postulated that, within cells, organic acids may electrostatically bind an equivalent quantity of cations and that amino acids may bind an equivalent quantity of both cations and anions.     

Neurofibromin GTPase-activating protein-related domains restore normal growth in Nf1-/- cells

Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21(Ras) (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primary Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function of NF1 GRD GAP activity on p21(Ras). Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types.     

Queuine-containing isoacceptor of tyrosine tRNA in Drosophila melanogaster: Alteration of levels by divalent cations

Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(?), ones of tRNA^Tyr, tRNA^His and tRNA^Asp of Drosophila melanogaster. Of the other divalent cations examined, Sr²⁺, Ni²⁺, Cu²⁺, Zn²⁺ and Hg²⁺, only Hg²⁺ failed to cause an increase in Q(+)tRNA^Tyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responsed to divalent ions; Hg²⁺ as well as Cd²⁺, Sr²⁺ and Zn²⁺ caused an increase in Q(+)tRNA^Tyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd²⁺-containing diet, they formed significantly more Q(+)tRNA^Tyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the ‘hard-soft’ classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(?) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation.     

Regulatory issues for commercialization of tomatoes with an antisense polygalacturonase gene

Summary Significant progress has been made in development of transgenic plants containing agriculturally useful genes. Concurrent with scientific advances has been development of a regulatory infrastructure within the U.S. Department of Agriculture (USDA) for assessing safety of controlled release of genetically engineered plants into the environment, as well as creation of a food policy by the Food and Drug Administration (FDA). Field trials and safety assessments of tomato containing an antisense polygalacturonase gene (FLAVR SAVR™ tomato) have been conducted. A detailed safety analysis of thekan ^r selectable marker was also done. Based on these data plus nutritional measurements, lack of changes in levels of natural toxins, and lack of any unintended changes, we have requested that the USDA and FDA determine that this genetically engineered tomato is safe for release into the environment and human consumption. Presented in the Session-in-Depth “Field Test Requirements and Performance of Transgenic Plants” at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Expression of IL-20 in synovium and lesional skin of patients with psoriatic arthritis: differential response to alefacept treatment

Introduction

Psoriatic arthritis (PsA) is an inflammatory joint disease associated with psoriasis. Alefacept (a lymphocyte function-associated antigen (LFA)-3 Ig fusion protein that binds to CD2 and functions as an antagonist to T-cell activation) has been shown to result in improvement in psoriasis but has limited effectiveness in PsA. Interleukin-20 (IL-20) is a key proinflammatory cytokine involved in the pathogenesis of psoriasis. The effects of alefacept treatment on IL-20 expression in the synovium of patients with psoriasis and PsA are currently unknown.

Methods

Eleven patients with active PsA and chronic plaque psoriasis were treated with alefacept (7.5 mg per week for 12 weeks) in an open-label study. Skin biopsies were taken before and after 1 and 6 weeks, whereas synovial biopsies were obtained before and 4 and 12 weeks after treatment. Synovial biopsies from patients with rheumatoid arthritis (RA) (n = 10) were used as disease controls. Immunohistochemical analysis was performed to detect IL-20 expression, and stained synovial tissue sections were evaluated with digital image analysis. Double staining was performed with IL-20 and CD68 (macrophages), and conversely with CD55 (fibroblast-like synoviocytes, FLSs) to determine the phenotype of IL-20-positive cells in PsA synovium. IL-20 expression in skin sections (n = 6) was analyzed semiquantitatively.

Results

IL-20 was abundantly expressed in both PsA and RA synovial tissues. In inflamed PsA synovium, CD68⁺ macrophages and CD55⁺ FLSs coexpressed IL-20, and its expression correlated with the numbers of FLSs. IL-20 expression in lesional skin of PsA patients decreased significantly (P = 0.04) 6 weeks after treatment and correlated positively with the Psoriasis Area and Severity Index (PASI). IL-20 expression in PsA synovium was not affected by alefacept.

Conclusions

Conceivably, the relatively limited effectiveness of alefacept in PsA patients (compared with anti-tumor necrosis factor (TNF) therapy) might be explained in part by persistent FLS-derived IL-20 expression.     

Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse ESCs introduced to suspension culture at a range of rotary orbital speeds (20–60 rpm) exhibited variable EB formation sizes and yields due to differences in the kinetics of cell aggregation. Computational fluid dynamic analyses indicated that rotary orbital shaking generated relatively uniform and mild shear stresses (≤2.5 dyn/cm²) within the regions EBs occupied in culture dishes, at each of the orbital speeds examined. The hydrodynamic conditions modulated EB structure, indicated by differences in the cellular organization and morphology of the spheroids. Compared to static culture, exposure to hydrodynamic conditions significantly altered the gene expression profile of EBs. Moreover, varying rotary orbital speeds differentially modulated the kinetic profile of gene expression and relative percentages of differentiated cell types. Overall, this study demonstrates that manipulation of hydrodynamic environments modulates ESC differentiation, thus providing a novel, scalable approach to integrate into the development of directed stem cell differentiation strategies. Biotechnol. Bioeng. 2010; 105: 611–626. © 2009 Wiley Periodicals, Inc.