Evaluation by double-blind placebo-controlled oral challenge of the clinical relevance of IgE antibodies against plant glycans.

BACKGROUND: The clinical relevance of immunoglobulin E (IgE) to plant glycans is a longstanding debate. We sought to evaluate their clinical reactivity using the human glycoprotein lactoferrin expressed in rice. METHODS: Allergic patients with IgE antibodies against plant glycans were analyzed for the presence of IgE against rice-produced lactoferrin. The potency of IgE to induce mediator release was assessed by basophil histamine release and skin prick tests (SPTs). Clinical relevance was evaluated by double-blind placebo-controlled oral challenge (DBPCOC). RESULTS: Twenty-four of 29 sera (82.7%) with IgE antibodies against plant glycans demonstrated IgE binding to transgenic lactoferrin. In three of five cases transgenic lactoferrin induced histamine release. Compared to a control major grass pollen allergen lactoferrin concentrations needed for biological activity of IgE were 5-6 orders of magnitude higher. Skin prick test and DBPCOC were negative in five patients with potential clinical reactivity that volunteered to undergo these in vivo challenges. CONCLUSIONS: Poor or no biological activity and lack of clinical relevance of IgE-binding plant glycans (five out of five) was demonstrated using human lactoferrin expressed in rice as a model.

28-day repeated dose oral toxicity of recombinant human holo-lactoferrin in rats.

Recombinant human holo-lactoferrin (holo-rhLF) was orally administered, via gavage, to Wistar rats at 1000, 500 and 100mg/kgbw/day for 28 days. The test article, holo-rhLF, was expressed in rice grain, extracted, purified and saturated with iron. During the 28-day period, animals were examined for evidence of toxicity. On day 29, the animals were exsanguinated, examined for gross pathology, and tissues preserved for histopathology. There were no deaths caused by holo-rhLF and in-life physical signs were generally normal. Although statistical differences were noted in some hematology, clinical chemistry and heart/body weight ratios, they were of questionable biological significance. A significantly greater total iron binding capacity (TIBC) was detected in the blood of male animals dosed with holo-rhLF. Serum was analyzed for the presence of IgG and IgE antibodies; demonstrating low levels of IgG antibodies to the human protein, but no increase in IgE antibodies. There was no increase in serum lactoferrin levels. The results of the 28-day oral administration demonstrate a lack of toxicity of holo-rhLF in rats. There were no treatment related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weights or pathology. The no observed adverse effect level (NOAEL) is greater than 1000 mg/kg/day.

Single-molecule conductance of redox molecules in electrochemical scanning tunneling microscopy.

Experimental data and theoretical notions are presented for 6-[1'-(6-mercapto-hexyl)-[4,4']bipyridinium]-hexane-1-thiol iodide (6V6) "wired" between a gold electrode surface and tip in an in situ scanning tunneling microscopy configuration. The viologen group can be used to "gate" charge transport across the molecular bridge through control of the electrochemical potential and consequently the redox state of the viologen moiety. This gating is theoretically considered within the framework of superexchange and coherent two-step notions for charge transport. It is shown here that the absence of a maximum in the Itunneling versus electrode potential relationship can be fitted by a "soft" gating concept. This arises from large configurational fluctuations of the molecular bridge linked to the gold contacts by flexible chains. This view is incorporated in a formalism that is well-suited for data analysis and reproduces in all important respects the 6V6 data for physically sound values of the appropriate parameters. This study demonstrates that fluctuations of isolated configurationally "soft" molecules can dominate charge transport patterns and that theoretical frameworks for compact monolayers may not be directly applied under such circumstances.

Neuropathological assessment of artemether-treated severe malaria.

In animals, high doses of intramuscular artemether and artemotil have been shown to cause an unusual pattern of selective damage to certain brainstem nuclei, especially those implicated in hearing and balance. We aimed to investigate whether a similar pattern arises in human adults. We examined the brainstems of adults who died after treatment with high dose artemether or quinine for severe falciparum malaria for evidence of a pattern of selective neuronal damage. Neuropathological findings were similar in recipients of quinine (n=15) and artemether (n=6; total artemether doses received 4-44 mg/kg). No evidence was recorded for artemether-induced neurotoxic effects.

Monoclonal antibodies to HIV-1 p24 core protein include pairs which exhibit synergistic binding.

Gamma and kappa chain cDNAs from four mouse monoclonal antibodies (mAbs) which bind three different sites on the core antigen (p24) of HIV-1 have been cloned and their V-region sequences determined. These mAbs are part of a larger group of seven anti-p24 mAbs analyzed in simultaneous competition assays with HIV-1 lysate as antigen and in protein blotting experiments using 10 carboxy-terminal truncations of a p24 fusion protein. One mAb, BB128, recognizes the p24 loop sequence EAAEWDRVHP and enhances the binding of two other mAbs (BI1777 and BI1279) when tested pairwise in simultaneous competition assays. The two monoclonals enhanced by BB128 recognize different antigenic sites on p24, with BI1777 binding to carboxy-terminal sequences and BI1279 to amino-terminal residues. In the pairwise assays, mAb BI1279 also acts as enhancing antibody for BI1777, as does mAb BB328, which recognizes residues in the central region of p24. Since aggregated p24 monomers form the HIV-1 capsid, p24 is a multivalent antigen in HIV-1 lysate. It seems likely, therefore, that synergistic binding of mAb pairs to p24 is effected by bivalent binding of the enhancing mAb stabilizing a conformation favorable for bivalent binding of the enhanced mAb.

Comparison of artemisinin suppositories, intramuscular artesunate and intravenous quinine for the treatment of severe childhood malaria.

Severe malaria remains a major cause of mortality and morbidity for children living in many tropical regions. With the emergence of strains of Plasmodium falciparum resistant to both chloroquine and quinine, alternative antimalarial agents are required. The artemisinin group of compounds are rapidly effective in severe disease when given by intramuscular or intravenous injection. However, these routes of administration are not always available in rural areas. In an open, randomized comparison 109 Vietnamese children, aged between 3 months and 14 years, with severe P.falciparum malaria, were allocated at random to receive artemisinin suppositories followed by mefloquine (n = 37), intramuscular artesunate followed by mefloquine (n = 37), or intravenous quinine followed by pyrimethamine/sulfadoxine (n = 35). There were 9 deaths: 2 artemisinin, 4 artesunate and 5 quinine-treated children. There was no difference in fever clearance time, coma recovery, or length of hospital stay among the 3 groups. However, parasite clearance times were significantly faster in artemisinin and artesunate-treated patients than in those who received quinine (P < 0.0001). Both artemisinin and artesunate were very well tolerated, but children receiving these drugs had lower peripheral reticulocyte counts by day 5 of treatment than those in the quinine group (P = 0.011). No other adverse effect or toxicity was found. There was no treatment failure in these 2 groups, but 4 patients in the quinine group failed to clear their parasites within 7 d of starting treatment and required alternative antimalarial therapy. Artemisinin suppositories are easy to administer, cheap, and very effective for treating children with severe malaria. In rural areas where medical facilities are lacking these drugs will allow antimalarial therapy to be instituted earlier in the course of the disease and may therefore save lives.

Pharmacokinetics of oral artesunate in children with moderately severe Plasmodium falciparum malaria.

The pharmacokinetic properties of oral artesunate (3 mg/kg) were determined in 10 Vietnamese children, aged from 6 to 15 years, with acute falciparum malaria of moderate severity. Plasma concentrations were measured using a bioassay and expressed in terms of antimalarial activity equivalent to dihydroartemisinin, the principal biologically active metabolite. Oral artesunate was absorbed rapidly with a mean time to peak plasma bioactivity of 1.7 h (95% confidence interval [95% CI] 0.8-2.6). There was wide variation in peak plasma concentrations with a mean value equivalent to 664 ng of dihydroartemisinin/mL (95% CI 387-9410, range 179-1395) and a four-fold variation in the area under the plasma concentration-time curves. Elimination from plasma was rapid with a mean (95% CI) half-life of 1.0 h (95% CI 0.8-1.4). Plasma antimalarial levels were below the limit of detection in all cases by 12 h, despite the relatively high dose of artesunate used. Oral artesunate is rapidly absorbed and rapidly eliminated in children with moderately severe malaria but there is considerable variation between individuals.

Electrocardiographic monitoring in severe falciparum malaria.

Electrocardiographic monitoring over 24 h was performed with 53 patients with severe Plasmodium falciparum malaria (11 adults and 42 children) to assess the frequency of unrecognized cardiac arrhythmias. Nine patients (17%) died, 5 during the monitoring period and 4 afterwards. Pauses lasting 2-3 s were observed in 3 children, a single couplet in one, and a further child experienced frequent supraventricular ectopic beats which had not been detected clinically. In none of the patients who died could death be attributed to cardiac arrhythmia. Furthermore, no abnormality was detected which could have resulted from the often large doses of quinine, chloroquine or the artemisinin derivatives used for treatment. These results suggest that the heart is remarkably resilient even in the face of heavy parasite sequestration and other vital organ dysfunction, and that deaths from cardiac arrhythmias in severe malaria are rare. The need for routine cardiac monitoring of patients with severe and complicated P. falciparum malaria is questionable.

28-Day repeated dose oral toxicity of recombinant human apo-lactoferrin or recombinant human lysozyme in rats.

Lactoferrin and lysozyme are important proteins of the human innate immune system. These proteins are found in breast milk and have been associated with improved infant health. Recombinant human apo-lactoferrin (apo-rhLF), 1800 and 180mg/kg bw/day, and recombinant human lysozyme (rhLZ), 360 and 36mg/kg bw/day, were orally administered to Wistar rats for 28 days. Apo-rhLF and rhLZ were expressed in rice grain, extracted, purified; the lactoferrin was iron desaturated. The animals were examined for evidence of toxicity; there were no deaths and in-life physical signs were normal. Transient differences in mean food consumption occurred in high dose apo-rhLF and low dose LZ females at week three. There were no biologically significant differences in hematological or clinical chemistry parameters. Necropsy results were normal and microscopic evaluation showed no treatment related changes in animals dosed with 1800mg/kg/day apo-rhLF or 360mg/kg/day rhLZ. The results of the 28-day oral administration demonstrate a lack of toxicity of apo-rhLF and rhLZ in rats. There were no treatment related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weight and pathology. The no observed adverse effect level (NOAEL) is greater than 1800mg/kg/day for apo-rhLF and 360mg/kg/day for rhLZ.

Bioactive recombinant human lactoferrin, derived from rice, stimulates mammalian cell growth.

Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (<10% iron saturation) or holo-rhLF (>90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [(3)H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.

Cerebral calpain in fatal falciparum malaria.

Disruption of axonal transport may represent a final common pathway leading to neurological dysfunction in cerebral malaria (CM). Calpains are calcium (Ca2+)-activated cysteine proteases which have been implicated in axonal injury in neurological diseases of various aetiologies. In this study we examined the association between mu- and m-calpain, the specific inhibitor calpastatin, and axonal injury in post mortem brain tissue from patients who died from severe malaria. Calpains were associated with axons labelled for the beta-amyloid precursor protein that detects impaired axonal transport. Elevated levels of calpastatin were rarely observed in injured axons. There were increased numbers of neurones with mu-calpain in the nuclear compartment in severe malaria cases compared with non-neurological controls, and increased numbers of glia with nuclear mu-calpain in CM patients compared with non-CM malaria cases and non-neurological controls. There was marked redistribution of calpastatin in the sequestered Plasmodium falciparum-infected erythrocytes. Responses specific to malaria infection were ascertained following analysis of brain samples from fatal cases with acute axonal injury, HIV encephalitis, and progressive multifocal leucoencephalopathy. Our findings implicate a role for calpains in the modulation of disease progression in CM.

Predicting the clinical outcome of tetanus: the tetanus severity score.

OBJECTIVES: To create a new tetanus score and compare it with the Phillips and Dakar scores. METHODS: We used prospectively acquired data from consecutive patients admitted to the Hospital for Tropical Diseases, Ho Chi Minh City, to create the Tetanus Severity Score (TSS) with multivariate logistic regression. We compared the new score with Phillips and Dakar scores by means of resubstituted and prospective data, assessing performance in terms of sensitivity, specificity and area under receiver operator characteristic curves. RESULTS: Resubstitution testing yielded a sensitivity of 77% (298/385) and a specificity of 82% (1,183/1,437) for the TSS; 89% (342/385) and 20% (281/1,437) for the Phillips score; and 13% (49/385) and 98% (1,415/1,437) for the Dakar score. The TSS showed greatest discrimination with 0.89 area under the receiver operator characteristic curve (95% CI 0.88-0.90); this was 0.74 for the Dakar score and (95% CI 0.71-0.77) and 0.66 for the Phillips score (95% CI 0.63-0.70; P values <0.001). Prospective testing showed 65% (13/20) sensitivity and 91% (210/230) specificity for the TSS; 80% (16/20) and 51% (118/230) for the Phillips score; and 25% (5/20) and 96% (221/230) for the Dakar score. The TSS achieved the greatest area under TSS of 0.89 (95% CI 0.82-0.96), significantly greater than the Phillips score [0.74 (0.6-0.88), P = 0.049] but not the Dakar score [0.80, (0.71-0.90), P = 0.090]. CONCLUSIONS: The TSS is the first prospectively developed classification scheme for tetanus and should be adopted to aid clinical triage and management and as a basis for clinical research.

Effects of diamines on ornithine decarboxylase activity in control and virally transformed mouse fibroblasts.

1. The induction of ornithine decarboxylase activity in mouse 3T3 fibroblasts or an SV-40 transformed 3T3 cell line by serum was prevented by addition of the naturally occurring polyamines putrescine (butane-1,4-diamine) and spermidine. Much higher concentrations of these amines were required to fully suppress ornithine decarboxylase activity in the transformed SV-3T3 cells than in the 3T3 fibroblasts. 2. Synthetic alpha omega-diamines with 3--12 carbon atoms also prevented the increase in ornithine decarboxylase activity induced by serum in these cells. The longer chain diamines were somewhat more potent than propane-1,3-diamine in this effect, but the synthetic diamines were less active than putrescine in the 3T3 cells. There was little difference between the responses of 3T3 and SV-3T3 cells to the synthetic diamines propane-1,3-diamine and heptane-1,7-diamine. 3. These results are discussed in relation to the control of polyamine synthesis in mammalian cells.

Prognostic value of electrocardiographic monitoring of patients with severe diphtheria.

The clinical, the 12-lead, and the 24-hour electrocardiographic findings in 15 consecutively studied Vietnamese children (aged 7 months to 16 years) with severe diphtheria were documented. Five patients died, three from respiratory arrest and two from cardiogenic shock; one of these two patients had complete heart block that necessitated insertion of a pacemaker. Electrocardiographic abnormalities were detected by 24-hour monitoring in all 15 cases, even though most patients had no clinical signs of myocarditis. Rates of supraventricular and ventricular ectopy were elevated and remained high long after other clinical manifestations were no longer noted. The degree of ventricular ectopy at the time of presentation was significantly associated with fatal outcome. In this series, more than two ventricular ectopic beats on a recording upon admission to the hospital predicted fatal outcome with 100% sensitivity and 100% specificity. A variety of nonsustained bradyarrhythmias and tachyarrhythmias were also observed up until discharge from the hospital. The time course of recovery from diphtheritic myocarditis is longer than has been appreciated previously.

Role of polyamines in differentiation of 3T3-L1 fibroblasts into adipocytes.

The role of polyamines in the differentiation of 3T3-L1 fibroblasts into adipose cells was studied. This conversion was blocked by the addition of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, which prevented a rise in spermidine content in the differentiating cells. The inhibition of differentiation could be overcome completely by the provision of exogenous putrescine, spermidine, or spermine. Partial reversal could be produced by exposure to nonphysiological homologues of the natural polyamines such as 1,3-diaminopropane, 1,5-diaminopentane, and sym-norspermine. Reversal of the inhibition of differentiation by exogenous polyamines required a period of exposure to the amines, indicating that the lack of differentiation is not due simply to an obligatory role for polyamines in the biosynthesis of lipids. These results indicate that spermidine is required for the differentiation, but spermidine alone was not able to replace insulin and 1-methyl-3-isobutylxanthine in stimulating conversion to adipocytes. Therefore spermidine appears to be necessary but not sufficient for differentiation to occur. Finally, the elevation of spermidine content that occurs during the conversion of fibroblasts to adipocytes did not correlate with an increased activity of the polyamine biosynthetic enzymes. This implies that the increase must be regulated by changes in the rate of degradation or excretion of the polyamines.

Regulation of polyamine content in cultured fibroblasts.

The content of putrescine and of the polyamines (spermidine and spermine) and the activities of their biosynthetic enzymes were measured in 3T3 mouse fibroblasts and SV40-transformed mouse fibroblasts over the entire period from subculturing in fresh medium until confluence. The transformed cells had a substantially higher content of putrescine and spermidine than the 3T3 cells and higher activities of all of the biosynthetic enzymes. However, the ratio of spermine synthase to spermidine synthase was higher in the 3T3 cells, which correlated with their higher spermine-to-spermidine ratio. All of the biosynthetic enzymes increased in activity during cell growth. Ornithine decarboxylase increased 20-fold with a maximum at 24-36 h after culturing whereas S-adenosylmethionine decarboxylase increased 3-fold at the same time. Spermidine synthase increased 10- to 16-fold during the growth period whereas spermine synthase increased 2- to 3-fold. The relative enzyme activities and the changes in total polyamine content suggested that 1) the activity of S-adenosylmethionine decarboxylase limited the production of the polyamines and 2) the relative amounts of spermidine and spermine synthase determined the predominant polyamine that the available decarboxylated S-adenosylmethionine is used to synthesize. When 3T3 cells become quiescent at confluence, there was a substantial fall in the intracellular spermidine level because of a greatly increased excretion of spermidine into the medium. Spermine content also fell because there was an increased conversion of spermine into spermidine, which was then excreted. The specific excretion of spermidine did not occur with the transformed SV-3T3 cells.