Anandamide prior to sensitization increases cell-mediated immunity in mice

The endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal organisms and capacity to play a modulatory role on several physiological systems, including modulation of immunity. Many studies have shown that administration of cannabinoids causes inhibitory effects on immune cells, including decreased proliferation and antigen-presenting cell (APC) costimulatory activity. In contrast, other groups have shown that some cannabinoids might present stimulatory actions on macrophage activity and T cell activation. Therefore, we aimed to investigate whether a treatment in vivo with a low dose of anandamide (0.1 mg/kg) immediately prior to sensitization would have an immunosuppressive or immunostimulatory effect on cell-mediated immunity (Th1 response) in mice. We report here that anandamide, prior to sensitization, was able to increase the Th1 response to ovalbumin in vivo and ex vivo. Anandamide increased delayed type hypersensitivity (DTH), splenocyte proliferation, and IFN-gamma production in a co-culture of adherent and non-adherent splenocytes. Moreover, anandamide prior to sensitization increased both the expression of DC co-stimulatory molecules (CD80/CD86) and IL-12/IL23 (p40) production ex vivo. We have also assessed direct effects of anandamide in the IFN-gamma/IL-4 balance of ConA-stimulated splenocytes in vitro. Anandamide at nanomolar concentrations increased the production of IFN-gamma, while such production decreased at micromolar range. Thus, anandamide induced both the increment of DC activation and IFN-gamma production, which are likely the mechanisms involved in the increase of Th1 response reported here. (C) 2010 Elsevier B.V. All rights reserved.

Occurrence of Sarcocystis falcatula in Captive Psittacine Birds in Brazil

Thirty-eight captive psittacine birds housed in a bird park in Foz do Iguacu, Parana, Brazil, died within a 15-month period as a result of infection with Sarcocystis falcatula. Although fatalities affected 16 species of psittacine birds, mortality was highest in Old World species, which were most susceptible to the pulmonary form of sarcocystosis. Along with the pathologic findings of this disease outbreak, a review of the pathophysiology of sarcosporidiosis is presented.

Antimicrobial Effects of Calcium Hydroxide and Chlorhexidine on Enterococcus faecalis

Introduction: Endodontic treatment is commonly based on nonspecific elimination of intraradicular micro-organisms. Although some authors prefer single-visit root canal operations for endodontic treatment, several studies have shown the importance of intracanal medication between sessions to kill microorganisms that biomechanical preparations alone cannot achieve. The purpose of this study was to evaluate the efficacy of calcium hydroxide Ca(OH)2 and chlorhexidine gel on the elimination of intratubular Enterococcus faecalis. Methods: Human uniradicular teeth contaminated with E. faecalis were treated with Ca(OH)(2), 2% chlorhexidine gel, Ca(OH)(2) plus 2% chlorhexidine gel, or saline (0.9% NaCl) as a negative control. Samples obtained at a depth of 0 to 100 mu m and 100 to 200 mu m from these root canal preparations were analyzed for bacterial load by counting the number of colonyforming units (CFUs) and bacterial viability using fluorescence microscopy. Results: A significant decrease in the number of CFUs and the percentage of viable E. faecalis was observed after treatment with either Ca(OH)(2) or chlorhexidine when compared with the control group. Additionally, chlorhexidine gel had a significantly higher antimicrobial efficacy as measured by the number of CFUs and the percentage of viable cells than Ca(OH)(2). No differences were observed between the antimicrobial properties of chlorhexidine gel with and without the addition of Ca(OH)(2). Conclusion: Both Ca(OH)(2) and chlorhexidine have antimicrobial effects on E. faecalis. Chlorhexidine had increased antimicrobial activity when compared with Ca(OH)(2.) Ca(OH)(2) combined with chlorhexidine showed similar antimicrobial activity to chlorhexidine alone. (J Endod 2010;36:1389-1393)

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

The effect of resource aggregation at different scales: Optimal foraging behavior of Cotesia rubecula

Resources can be aggregated both within and between patches. In this article, we examine how aggregation at these different scales influences the behavior and performance of foragers. We developed an optimal foraging model of the foraging behavior of the parasitoid wasp Cotesia rubecula parasitizing the larvae of the cabbage butterfly Pieris rapae. The optimal behavior was found using stochastic dynamic programming. The most interesting and novel result is that the effect of resource aggregation within and between patches depends on the degree of aggregation both within and between patches as well as on the local host density in the occupied patch, but lifetime reproductive success depends only on aggregation within patches. Our findings have profound implications for the way in which we measure heterogeneity at different scales and model the response of organisms to spatial heterogeneity.

Physiological roles and regulation of mammalian sulfate transporters

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients; in cells and is the fourth most abundant anion in human plasma (300 muM). Sulfate is the major sulfur source in many organisms, and because it is a hydrophilic anion that cannot passively cross the lipid bilayer of cell membranes, all cells require a mechanism for sulfate influx and efflux to ensure an optimal supply of sulfate in the body. The class of proteins involved in moving sulfate into or out of cells is called sulfate transporters. To date, numerous sulfate transporters have been identified in tissues and cells from many origins. These include the renal sulfate transporters NaSi-1 and sat-1, the ubiquitously expressed diastrophic dysplasia sulfate transporter DTDST, the intestinal sulfate transporter DRA that is linked to congenital chloride diarrhea, and the erythrocyte anion exchanger AE1. These transporters have only been isolated in the last 10-15 years, and their physiological roles and contributions to body sulfate homeostasis are just now beginning to be determined. This review focuses on the structural and functional properties of mammalian sulfate transporters and highlights some of regulatory mechanisms that control their expression in vivo, under normal physiological and pathophysiological states.

Immune response to Bacteroides forsythus in a murine model

A murine skin abscess model was used to study the immune response to an acute infection with Bacteroides forsythus. BALB/c mice were given subcutaneous injections of either viable or heat-killed B. forsythus, while a third sham-immunized control group received phosphate-buffered saline. Weights and lesion sizes were measured. Blood was collected from the heart and specific antibodies to B. forsythus measured by an ELISA. Swabs taken from the lesions and also from pooled blood were cultured anaerobically for viable B. forsythus. Viable B. forsythus-induced lesions reached maximum size at day 7. B. forsythus cells were recovered from lesions up to day 4 although none were cultured from blood samples. Heat-killed bacteria induced much smaller lesions. Serum antibody levels increased during the 9-day study period, being significantly higher in mice injected with viable compared with heat-killed B. forsythus. Antibody levels in sham control mice were significantly lower than those seen in the other two groups. These results showed that a subcutaneous injection of viable cells of B. forsythus elicited a pronounced abscess formation and induce higher levels of specific antibodies compared with that produced by an injection of dead bacteria. This suggests that, as with other periodontopathic organisms, this mouse model can be used to study the immune response to B. forsythus.

Distribution of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in an Australian population

Background, aim: The present study describes (i) the natural distribution of the three putative periodontopathogens Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in an Australian population and (ii) the relationship between these organisms, pocket depths and supragingival plaque scores. Methods: Subgingival plaque was collected from the shallowest and deepest probing site in each sextant of the dentition. In total, 6030 subgingival plaque samples were collected from 504 subjects. An ELISA utilising pathogen-specific monoclonal antibodies was used to quantitate bacterial numbers. Results:: A. actinomycetemcomitans was the most frequently detected organism (22.8% of subjects) followed by P. gingivalis and P. intermedia (14.7% and 9.5% of subjects respectively). The majority of infected subjects (83%) were colonised by a single species of organism. A. actinomyceteincomitans presence was overrepresented in the youngest age group but under-represented in the older age groups. Conversely, P. gingivalis and P. intermedia presence was under-represented in the youngest age group but over-represented in the older age groups. Differing trends in the distribution of these bacteria were observed between subjects depending upon the site of the infection or whether a single or mixed infection was present; however, these differences did not reach significance. Bacterial presence was strongly associated with pocket depth for both A. actinomyceteincomitans and P. gingivalis. For A. actinomycetemcomitans, the odds of a site containing this bacterium decrease with deeper pockets. In contrast, for P. gingivalis the odds of a site being positive are almost six times greater for pockets >3 ram than for pockets less than or equal to3 nun. These odds increase further to 15.3 for pockets deeper than 5 mm. The odds of a site being P. intermedia positive were marginally greater (1.16) for pockets deeper than 3 mm. Conclusions: This cross-sectional study in a volunteer Australian population, demonstrated recognised periodontal pathogens occur as part of the flora of the subgingival plaque. Prospective longitudinal studies are needed to examine the positive relationship between pocket depth and pathogen presence with periodontal disease initiation and/or progression.

Chryseobacterium in burn wounds

Chryseobacteria are gram negative organisms, formerly known as Flavobacteria, which rarely cause infections of burn wounds. This article documents three casts of Chryseobacterium infection in burn wounds and adds to the other two cases that have been reported in English literature. Two patients died, with one of the deaths linked to a Chryseobacteria bacteraemia. In two patients, there was an associated history of first aid treatment with untreated water. Patients whose burn wounds are suspected to be infected with Chryseobacterium require wound excision and coverage in combination with antibiotic therapy such as ciprofloxacin, vancomycin and rifampicin. (C) 2001 Elsevier Science Ltd and ISBI. All rights reserved.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular Information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability In three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced similar to 50 scoreable polymorphic DNA markers, between individuals of three Independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from Individual DNA samples that had been combined to create the bulked samples.

ENCORE: The effect of nutrient enrichment on coral reefs. Synthesis of results and conclusions

Coral reef degradation resulting from nutrient enrichment of coastal waters is of increasing global concern. Although effects of nutrients on coral reef organisms have been demonstrated in the laboratory, there is little direct evidence of nutrient effects on coral reef biota in situ. The ENCORE experiment investigated responses of coral reef organisms and processes to controlled additions of dissolved inorganic nitrogen (N) and/or phosphorus (P) on an offshore reef(One Tree Island) at the southern end of the Great Barrier Reef, Australia. A multi-disciplinary team assessed a variety of factors focusing on nutrient dynamics and biotic responses. A controlled and replicated experiment was conducted over two years using twelve small patch reefs ponded at low tide by a coral rim. Treatments included three control reefs (no nutrient addition) and three + N reefs (NH4Cl added), three + P reefs (KH2PO4 added), and three + N + P reefs. Nutrients were added as pulses at each low tide (ca twice per day) by remotely operated units. There were two phases of nutrient additions. During the initial, low-loading phase of the experiment nutrient pulses (mean dose = 11.5 muM NH4+; 2.3 muM PO4-3) rapidly declined, reaching near-background levels (mean = 0.9 muM NH4+; 0.5 muM PO4-3) within 2-3 h. A variety of biotic processes, assessed over a year during this initial nutrient loading phase, were not significantly affected, with the exception of coral reproduction, which was affected in all nutrient treatments. In Acropora longicyathus and A. aspera, fewer successfully developed embryos were formed, and in A. longicyathus fertilization rates and lipid levels decreased. In the second, high-loading, phase of ENCORE an increased nutrient dosage (mean dose = 36.2 muM NH4+; 5.1 muM PO4-3 declining to means of 11.3 muM NH4+ and 2.4 muM PO4-3 at the end of low tide) was used for a further year, and a variety of significant biotic responses occurred. Encrusting algae incorporated virtually none of the added nutrients. Organisms containing endosymbiotic zooxanthellae (corals and giant clams) assimilated dissolved nutrients rapidly and were responsive to added nutrients. Coral mortality, not detected during the initial low-loading phase, became evident with increased nutrient dosage, particularly in Pocillopora damicornis. Nitrogen additions stunted coral growth, and phosphorus additions had a variable effect. Coral calcification rate and linear extension increased in the presence of added phosphorus but skeletal density was reduced, making corals more susceptible to breakage. Settlement of all coral larvae was reduced in nitrogen treatments, yet settlement of larvae from brooded species was enhanced in phosphorus treatments. Recruitment of stomatopods, benthic crustaceans living in coral rubble, was reduced in nitrogen and nitrogen plus phosphorus treatments. Grazing rates and reproductive effort of various fish species were not affected by the nutrient treatments. Microbial nitrogen transformations in sediments,were responsive to nutrient loading with nitrogen fixation significantly increased in phosphorus treatments and denitrification increased in all treatments to which nitrogen had been added. Rates of bioerosion and grazing showed no significant effects of added nutrients, ENCORE has shown that reef organisms and processes investigated ill situ were impacted by elevated nutrients. Impacts mere dependent on dose level, whether nitrogen and/or phosphorus mere elevated and were often species-specific. The impacts were generally sub-lethal and subtle and the treated reefs at the end of the experiment mere visually similar to control reefs. Rapid nutrient uptake indicates that nutrient concentrations alone are not adequate to assess nutrient condition of reefs. Sensitive and quantifiable biological indicators need to be developed for coral reef ecosystems. The potential bioindicators identified in ENCORE should be tested in future research on coral reef/nutrient interactions. Synergistic and cumulative effects of elevated nutrients and other environmental parameters, comparative studies of intact vs. disturbed reefs, offshore vs, inshore reefs, or the ability of a nutrient-stressed reef to respond to natural disturbances require elucidation. An expanded understanding of coral reef responses to anthropogenic impacts is necessary, particularly regarding the subtle, sub-lethal effects detected in the ENCORE studies. (C) 2001 Published by Elsevier Science Ltd.

A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data

The 16S rRNA gene (16S rDNA) is currently the most widely used gene for estimating the evolutionary history of prokaryotes, To date, there are more than 30 000 16S rDNA sequences available from the core databases, GenBank, EMBL and DDBJ, This great number may cause a dilemma when composing datasets for phylogenetic analysis, since the choice and number of reference organisms are known to affect the resulting tree topology. A group of sequences appearing monophyletic in one dataset may not be so in another. This can be especially problematic when establishing the relationships of distantly related sequences at the division (phylum) level. In this study, a multiple-outgroup approach to resolving division-level phylogenetic relationships is suggested using 16S rDNA data. The approach is illustrated by two case studies concerning the monophyly of two recently proposed bacterial divisions, OP9 and OP10.

Advances in the diagnosis of primary immunodeficiency disorders in childhood

Primary immunodeficiency disorders in childhood usually present as unusual, recurrent or severe infections, symptomatic infections with organisms of low pathogenicity, or as recognizable syndromes which are known to have associated immunological abnormalities. In many of the primary immunodeficiency disorders, there are known patterns of inheritance, and other family members may be affected. Some primary immunodeficiency disorders are relatively common, such as selective IgA deficiency, and often do not lead to major morbidity. Others, such as the severe combined immune deficiency syndromes, are relatively rare, and are fatal in early life if not recognized and treated early. Diagnosis of a primary immunodeficiency disorder depends on appropriate use of laboratory investigations. Often there will be abnormalities detected on a complete blood film and measurement of immunoglobulin isotypes. More complex investigations should be undertaken in conjunction with a paediatric immunology service. In recent years, many of the clinically defined primary immunodeficiency disorders have been shown to have associated specific gene defects. For some, this has led to the identification and characterization of defective or absent gene products. The consequences of this new knowledge are more accurate diagnosis, early diagnosis including antenatal diagnosis, detection of undiagnosed disease in other family members, and the potential for new therapies including gene or gene product therapy.

Drug targets and mechanisms of resistance in the anaerobic protozoa

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated ruing laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Drug susceptibility testing of anaerobic protozoa

A simple technique for routine, reproducible global surveillance of the drug susceptibility status of the anaerobic protozoa Trichomonas, Entamoeba, and Giardia is described, Data collected using this technique can be readily compared among different laboratories and with previously reported data. The technique employs a commercially available sachet and bag system to generate a low-oxygen environment and log, drug dilutions in microtiter plates, which can be monitored without aerobic exposure, to assay drug-resistant laboratory lines and clinically resistant isolates. MICs (after 2 days) of 3.2 and 25 muM indicated metronidazole-sensitive and highly clinically resistant isolates of T. vaginalis in anaerobic assays, respectively. The aerobic MICs were 25 and > 200 muM. MICs (1 day) of 12.5 to 25 muM were found for axenic lines of E. histolytica, and MICs for G. duodenalis (3 days) ranged from 6.3 muM for metronidazole-sensitive isolates to 50 muM for laboratory metronidazole-resistant lines. This technique should encourage more extensive monitoring of drug resistance in these organisms.