The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.     

Nitrogen requirement of the omnivorous greater bilby, Macrotis lagotis (Marsupialia: Peramelidae)

Mainly due to their utilisation of relatively low-fiber diets compared to herbivorous mammals, omnivores are expected to have correspondingly low maintenance nitrogen requirements (MNRs). The limited studies examining nitrogen requirements of omnivorous mammals to date have shown this to be the case. In this article, we determine the dietary MNR of greater bilbies (Macrotis lagotis), arid-zone omnivorous marsupials, by feeding them varying proportions of mixed seeds and dried currants (sun-dried grapes). We also examine the possibility that bilbies conserve nitrogen by recycling endogenous urea to their gastrointestinal tract. The dietary MNR of 127 mg N kg(-0.75) d(-1) calculated for the bilby falls within the range calculated for other marsupial omnivores and is lower than that of any of the herbivores. This low requirement for nitrogen was correlated with significant recycling of endogenous urea to the gut; bilbies recycled between 44% and 80% of urea synthesised in the liver, but the proportion recycled was independent of dietary nitrogen intake. The relatively low MNR of the bilby is consistent with its low rates of basal metabolism and the recycling of endogenous urea to the gut, both of which reduce urinary nitrogen losses, and a low-fiber diet that minimises metabolic fecal nitrogen loss. Because the bilby inhabits an environment where its food supply is unpredictable and of seasonally low nitrogen content, this species is likely to benefit from its low requirement for nitrogen.     

In vitro inhibition of Salmonella enterica serovars choleraesuis and typhimurium,Escherichia coli F-18, and Escherichia coli O157:H7 by a porcine continuous-flow competitive exclusion culture

A competitive exclusion (CE) culture of porcine cecal bacteria was developed as a continuous-flow culture in chemostats, was designated RPCF, and was used as a model to determine its usefulness against in vitro colonization by Salmonella enterica serovars Typhimurium and Choleraesuis, Escherichia coli strain F-18, and E. coli serotype O157:H7 (933). Chemostats with or without RPCF were inoculated with 10⁶ colony-forming units (CFU)/ml of Typhimurium, Choleraesuis, F-18, or O157:H7. Chemostats were sampled for salmonellae and E. coli at 15 min, 7 h, and every 24 h thereafter. In control chemostats without RPCF, Typhimurium, Choleraesuis, F-18, and O157:H7 rapidly established colonization and had concentrations of 10⁶ CFU/ml for 96–120 h post-inoculation. In the chemostats that contained RPCF, reductions (P < 0.05) of Choleraesuis, F-18, and O157:H7 were observed at 24 h post-inoculation. Typhimurium was decreased (P < 0.05) at 48 h post-inoculation, and by 120 h post-inoculation, all chemostats were negative for the four challenge microorganisms. These results demonstrate that RPCF cultures were able to inhibit the growth of Typhimurium, Choleraesuis, and E. coli strains F-18 and O157:H7 in vitro and suggest the potential for the use of CE in swine to prevent disease induced by these microorganisms. Received: 2 October 2001 / Accepted: 31 December 2001     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.     

Structural alterations of phospholipid film domain morphology induced by cholesterol

Structures of the monolayer films of dipalmitoylphosphatidylcholine (DPPC) mixed with different amounts of cholesterol were studied at air-water interface using surface pressure-area measurements, epifluorescence microscopy and atomic force microscopy (AFM). Pure DPPC, cholesterol or DPPC-cholesterol mixtures were dissolved in organic solvents with a small amount of fluorescently labeled phospholipid probe (NBD-PC) and spread onto the air-water interface. Surface pressure-area isotherms and epifluorescence microscopy of such films at the air-water interface suggested that DPPC undergoes a gas to fluid to condensed phase transition, while cholesterol undergoes a gas to solid-like transition. A shift of the surface pressure-area curve to lower area per molecule was observed when cholesterol was mixed with DPPC. Epifluorescence microscopy showed the formation of spiral shaped domains for mixed monolayers. Increase in cholesterol content abolished domain characteristics possibly due to fluidizing property of cholesterol. AFM measurements of monolayers, transferred onto freshly cleaved mica by Langmuir-Blodgett technique, revealed the alterations caused by cholesterol on the gel and fluid domains of such films. AFM measurements re-established similar trend in domain characteristics as evidenced in epifluorescence microscopy.     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Prevention of diseases caused by Staphylococcus aureus using the peptide RIP

Staphylococcus aureus causes many diseases including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. The heptapeptide RIP has been shown to prevent cellulitis in mice, which was induced by S. aureus strain Smith diffuse. Here we show that RIP can also significantly reduce the overall pathology and delay the onset of disease symptoms in several other models of S. aureus infections, including: keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) and septic arthritis (tested in mice against S. aureus LS-1). These findings substantiate that RIP is not strain specific in its inhibitory activity and that RIP is an effective inhibitor of bacterial pathology at multiple body sites following diverse routes and doses of administration. These findings strongly evidence the potential value of RIP as a chemotherapeutic agent.