Modulation of serum uric acid levels by inosine in patients with multiple sclerosis does not affect blood pressure.

The role of uric acid (UA) in human physiology is subject to controversy. Either it is an important radical scavenger, a mostly neutral, waste metabolic product that may cause gout and kidney stones if elevated, or it is involved in the causation of hypertension, vascular and renal diseases. Recently we conducted a clinical trial to determine whether raising the serum UA levels through the oral administration of inosine is well tolerated and may benefit patients with multiple sclerosis. An important aspect of the safety profile is whether raising the serum UA levels elevates blood pressure. During the 1-year trial, blood pressure and serum UA levels were monitored in 16 patients. Both parameters were recorded throughout the trial that included 69 visits by patients at baseline and during the placebo phase as well as 138 visits while receiving inosine treatment. We have observed that although the serum UA levels increased significantly during the inosine treatment phase of the trial, from 4.2+/-0.8 to 7.1+/-1.7 mg per 100 ml, blood pressure remained unchanged, averaging 123+/-15/78+/-9. Our findings indicate that raising the serum UA levels to upper normal physiological levels for a period of up to 1-year does not influence blood pressure significantly.

Production of recombinant anthrax toxin receptor (ATR/CMG2) fused with human Fc in planta.

Mass vaccination against anthrax with existing vaccines is costly and unsafe due to potential side effects. For post-infection treatment, passive immunotherapy measures are currently available, most based on anthrax protective antigen (PA)-specific therapeutic antibodies. Efficient against wild-type strains, these treatment(s) might fail to protect against infections caused by genetically engineered Bacillus anthracis strains. A recent discovery revealed that the von Willebrand factor A (VWA) domain of human capillary morphogenesis protein 2 (CMG2) is an exceptionally effective anthrax toxin receptor (ATR) proficient in helping to resolve this issue. Here we describe in planta production of chimeric recombinant protein (immunoadhesin) comprised of functional ATR domain fused with the human immunoglobulin Fc fragment (pATR-Fc). The fusion design allowed us to obtain pATR-Fc in plant green tissues in a soluble form making it fairly easy to purify by Protein-A chromatography. Standardized pATR-Fc preparations (purity>90%) were shown to efficiently bind anthrax PA as demonstrated by ELISA and Western blot analysis. Recombinant pATR-Fc was also shown to protect J774A1 macrophage cells against the anthrax toxin. This study confirmed that plant-derived pATR-Fc antibody-like protein is a prospective candidate for anthrax immunotherapy.

Rabies in the face of the 21st century.

Reference to an ancient Hindu picture of a snarling dog may be a convincing enough proof to consider the fact that rabies has been known in the world for the past 50 centuries. Prior to the monumental observation about rabies of Fracastoro in the 16th century, facts and fantasies were intermingled in the study of rabies. In the realm of fantasy, consider the statement of Aristotle (otherwise a great philosopher) that only animals and not humans die of rabies. It took 19 centuries before Fracastoro finally established that infection with rabies is lethal for all warm-blooded beings including humans. The new era of rabies dates from the time of Galtier who isolated the virus and Pasteur who was able to create a somewhat attenuated strain of virus fixe which became the tool of laboratory studies for many decades after Pasteur. During the last 50 years of the past century, our knowledge of rabies increased by leaps and bounds. First of all, using molecular biology as a tool, it was possible to 'take the virus apart', so to speak and describe and analyse all of its components. Establishment of multivariability among viruses as 'de la rue' permitted not only a construct of a genetic linkage among lyssa family but also solved some puzzles of pathogenesis of rabies which defied solution when all work concentrated on one laboratory strain of the virus. As an example, we know much more now about the genetic background regulating virulence of the virus. In addition, it is now possible to use rabies as a vector of biological materials such as vaccines or sera. There is no progress in the treatment of the uniformly lethal disease. Perhaps, optimistically speaking, the 21st century will bring us a glimmer of hope for the successful treatment of human rabies.

Generation of plant-derived recombinant DTP subunit vaccine.

The current diphtheria-tetanus-pertussis (DTP) pediatric vaccine is produced from the corresponding pathogenic bacteria Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis; five injected doses of DTaP (acellular) vaccine are required for every child in the standard US vaccination schedule. Because the vaccine is derived from native live sources, adverse effects are possible and production is complex and costly. To address issues of safety, ease of renewability and expense, we used recombinant technology in an effort to develop a subunit DPT vaccine derived in non-pathogenic plant expression systems. Expression of diphtheria toxin (DT), tetanus fragment-C (TetC) and the non-toxic S1 subunit of pertussis toxin (PTX S1) antigenic proteins in soluble form in low-alkaloid tobacco plants and carrot cell cultures allowed efficient downstream purification to levels suitable for intramuscular injection in BALB/c mice. At working concentrations of 5mug per dose, these preparations induced high levels of antigen-specific IgGs in mouse sera. Our results clearly support the feasibility of producing recombinant pediatric vaccine components in plants.

Immunological assessment of plant-derived avian flu H5/HA1 variants.

Polypeptide variants of the HA1 antigenic domain of the H5N1 avian influenza virus hemagglutinin (HA) molecule were produced in plants using transient and stable expression systems and fused with His/c-myc tags or with mouse or human Fc antibody fragments. The resulting peptides were purified and used for intramuscular immunization of mice. While the recombinant HA1 variants induced a significant serum humoral immune response in the mice, none of the HA1 preparations induced virus-neutralizing antibodies. Fusion with the Fc fragment improved overall yield of the constructs and allowed purification requiring only a single step, but led to no detectable fusion-related enhancement of immunogenicity or quality of immune response.

Role of uric acid in multiple sclerosis.

In the past decade, a growing number of evidence has implicated free radicals in a variety of pathophysiological conditions including aging, cancer, and coronary heart disease. Analyses of different aspects of multiple sclerosis (MS) pathology with respect to oxidative damage have also revealed evidence of free radical injury to the central nervous system (CNS), although attempts to protect the CNS using various antioxidants have met with only moderate success. Several recent studies have reported lower levels of uric acid (UA), a major scavenger of reactive nitrogen species, in MS patients, while other studies found no such correlation. Here, we discuss these studies as well as current efforts to manipulate serum UA levels in MS patients.

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta.

Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.

Pathogenesis of rabies.

Rabies is a central nervous system (CNS) disease that is almost invariably fatal. The causative agent is rabies virus (RV), a negative-stranded RNA virus of the rhabdovirus family. RV pathogenesis, like that of other viruses, is a multigenic trait. Recent findings indicate that in addition to the RV G protein viral elements that regulate gene expression, especially expression of the L gene, are also likely to play a role in RV pathogenesis. In vivo, RV infects almost exclusively neurons, and neuroinvasiveness is the major defining characteristic of a classical RV infection. A key factor in the neuroinvasion of RV is transsynaptic neuronal spread. While the ability of RV to spread from the post-synaptic site to the pre-synaptic site is mediated by the RV G protein, the RV P protein might be an important determinant of retrograde transport of the virus within axons. Although the mechanism(s) by which an RV infection cause(s) a lethal neurological disease are still not well understood, the most significant factor underlying the lethal outcome of an RV infection appears to be the neuronal dysfunction due to drastically inhibited synthesis of proteins required in maintaining neuronal functions.

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine.

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.

Uric acid levels in patients with multiple sclerosis: analysis in mono- and dizygotic twins.

Presence of nitrotyrosine in cells surrounding plaques indicates that peroxynitrite may be the cause of brain lesions in multiple sclerosis. Low levels of uric acid, a natural scavenger of peroxynitrite, were demonstrated in blood of patients with multiple sclerosis in comparison with control individuals. These observations were now extended to 132 sets of twins with one sibling affected by multiple sclerosis. In blood of both mono- and dizygotic twins the uric acid levels were lower in the twin with the disease than in the healthy twin.

Hypotheses and facts.

The book, The river, is based on assumptions and not facts. Oral polio vaccine was produced entirely in rhesus monkey kidney cell cultures. Allegations that it was produced in chimpanzee kidneys at the Wistar Institute in Philadelphia or, alternatively, that the vaccine was made in the then Belgian Congo in chimpanzee kidney has no basis in fact. As the only witness to the historical events leading to the development of oral polio vaccine, I have demonstrated in this paper the truthful facts excluding any link between oral polio vaccine and human immunodeficiency virus.

Experimental utility of rabies virus-neutralizing human monoclonal antibodies in post-exposure prophylaxis.

Rabies immune globulin (RIG) is essential for post-exposure prophylaxis but is expensive and not widely available. Rabies virus-neutralizing human monoclonal antibodies (Mabs) were evaluated in vitro and in a Syrian hamster model as a potential future alternative. Seven Mabs neutralized representative rabies virus variants. However, a European bat lyssavirus was not neutralized by either Mabs or RIG. Moreover, Duvenhage virus was neutralized by RIG, but not by Mabs, and Lagos bat and Mokola viruses were neutralized by one Mab but not by RIG. In hamsters, one Mab resulted in protection that was comparable to human RIG. These results suggest that Mabs may provide a promising alternative to RIG.

The green revolution: plants as heterologous expression vectors.

Agrobacterium tumefaciens mediated gene transfer into the plant genome laid the groundwork for new procedures aimed at crop improvement, including resistance to pathogens, increased product yield, modified oil content, and resistance to environmental stress conditions. New developments in molecular plant virology have led to the generation of plant-based systems for transient expression of foreign sequences using plant virus vectors. In the last decade both transgenic plants and plant virus vectors have been used increasingly to produce a wide range of biomedical reagents, including vaccine antigens, in a safe and economically feasible manner. These new plant-based technologies have enormous potential for a variety of applications, including the oral delivery of vaccine antigens.

Effect of rabies virus infection on gene expression in mouse brain.

A variety of molecular genetic approaches were used to study the effect of rabies virus (RV) infection on host gene expression in mouse brain. The down-regulation of gene expression was found to be a major effect of RV infection by using subtraction hybridization. However, a combination of techniques identified approximately 39 genes activated by infection. These included genes involved in regulation of cell metabolism, protein synthesis, synaptic activity, and cell growth and differentiation. Northern blot analysis to monitor temporal activation of several of these genes following infection revealed essentially two patterns of activation: (i) an early response with up-regulation beginning within 3 days after infection and correlating with transcription of RV nuclear protein; and (ii) a late response with enhanced expression occurring at days 6-7 after infection and associated with peak RV replication. The gene activation patterns and the known functions of their products suggest that a number of host genes may be involved in the replication and spread of RV in the brain.

Pathogenesis of Alfalfa mosaic virus in Soybean (Glycine max) and Expression of Chimeric Rabies Peptide in Virus-Infected Soybean Plants.

ABSTRACT Infection of soybean (Glycine max) plants inoculated with particles of Alfalfa mosaic virus (AlMV) isolate 425 at 12 days after germination was monitored throughout the life cycle of the plant (vegetative growth, flowering, seed formation, and seed maturation) by western blot analysis of tissue samples. At 8 to 10 days after inoculation, the upper uninoculated leaves showed symptoms of virus infection and accumulation of viral coat protein (CP). Virus CP was detectable in leaves, stem, roots, seedpods, and seed coat up to 45 days postinoculation (dpi), but only in the seedpod and seed coat at 65 dpi. No virus accumulation was detected in embryos and cotyledons at any time during infection, and no seed transmission of virus was observed. Soybean plants inoculated with recombinant AlMV passaged from upper uninoculated leaves of infected plants showed accumulation of full-length chimeric AlMV CP containing rabies antigen in systemically infected leaves and seed coat. These results suggest the potential usefulness of plants and plant viruses as vehicles for producing proteins of biomedical importance in a safe and inexpensive manner. Moreover, even the soybean seed coat, treated as waste tissue during conventional processing for oil and other products, may be utilized for the expression of value-added proteins.