Presence of <Emphasis Type="Italic">C. albidus</Emphasis>, <Emphasis Type="Italic">C. laurentii</Emphasis> and <Emphasis Type="Italic">C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type="Italic">Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.     

Temperature influences the expression of fimbriae and flagella in <Emphasis Type="Italic">Hafnia alvei</Emphasis> strains: an immunofluorescence study

Hafnia alvei, a Gram negative bacillus related to the Enterobacteriaceae family, is considered an opportunistic pathogen of several animal species and humans. In this communication, we describe fimbrial-like structures from different strains of H. alvei that cannot be easily ascribed to any of the previously reported fimbrial types in this species (type I or type III). Polymerase chain reaction (PCR) and immunofluorescence assays were carried out to study fimbriae and flagella in H. alvei strains isolated from different sources. No correlation between the results obtained by PCR and those obtained by phenotypic methods were found, and the antibodies used gave cross or different recognition patterns of the surface structures present in these strains. We report as well that strain and growth temperature influence fimbriation and expression of flagella in human and animal isolates of H. alvei. This study also indicates that the absence of fimbriae have a significant positive influence on the initial adhesion of H. alvei to human epithelial cells.     

Anti-striated muscle antibody activity produced by Trypanosoma cruzi

We have previously shown that Trypanosoma cruzi shares antigenic determinants with preparations of the calcium-sequestering adenosine triphosphatase of sarcoplasmic reticulum. The cross-reacting antigen (SRA) is also apparently present on the sarcolemma of cardiac myofibers. Using highly specific reference antisera to either the small membranes of T. cruzi or to a tryptic fragment of striated muscle SRA, it was shown that SRA is present in the striated muscle of animals representative of the evolutionary scale ranging from nonhuman primate to fish. The small membranes of nine different T. cruzi strains isolated from widely divergent areas of the American continents also reacted with the reference antisera. This indicates that SRA is present in these T. cruzi strains and may be prevalent among all T. cruzi strains. The shared T. cruzi-striated muscle antigen, SRA, may be a heteroantigen present in all T. cruzi strains and in the striated muscle of all classes of animals. Immunization of rabbits (three of five) or chickens (five pairs of five pairs) with striated muscle membrane preparations of different classes of animals, particularly those of nonhuman primate, chicken, and turtle, gave rise to IgG anti-allogeneic striated muscle antibody activity. Immunization of rabbits (four of nine) and chickens (five pairs of six pairs) with the small membranes of different T. cruzi strains also produced IgG anti-allogeneic striated muscle. These data indicate that T. cruzi shares cross-immunogenicity with striated muscle SRA. Since SRA is apparently present on the sarcolemma of cardiac myofibers, it may be implicated in the immunopathogenesis of Chagas' disease.     

1-Aminocyclopropane-1-carboxylic acid as a substrate of peroxidase: conditions for oxygen consumption, hydroperoxide generation and ethylene production

Conditions in which 1-aminocyclopropane-1-carboxylic acid (ACC) functions as a substrate of peroxidase have been investigated by measuring oxygen consumption in the reaction medium and the production of ethylene. In both cases, the presence of Mn2+ and either H2O2 or the activated form of peroxidase, namely compound I of peroxidase, was found to be essential. Both oxygen consumption and ethylene production were dependent on enzyme concentration, the optimum ACC/Mn2+ ratio being 1:1. Oxygen consumption in a system with ACC, Mn2+ and compound I showed an enzyme-dependent lag phase and then proceeded to total depletion, suggesting that the system itself generates hydroperoxides that completed the catalytic cycle of the enzyme. The presence of these hydroperoxides in the reaction medium was detected by a colorimetric method. High H2O2 concentration progressively decreased oxygen consumption, the same effect being produced by catalase. Ethylene production was oxygen dependent, mediated by ACC-free radicals and gave a poor yield. The results suggest that the fate of these ACC-free radicals determines the yield in ethylene. These radicals must be oxidized immediately, otherwise their stabilization to hydroperoxides would prevent ethylene production.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

In vitro and in vivo characterization of an interleukin‐15 antagonist peptide by metabolic stability, 99mTc‐labeling,and biological activity assays

Interleukin (IL)–15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL‐15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with ^99mTc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL‐2 cells, using 3 different additives to improve the solubility of these peptides. The half‐life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with ^99mTc showed a yield of approximately 99.8%. The ^99mTc‐labeled peptide was stable in a 30‐fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL‐15 overexpression.     

Stringency in the absence of ppGpp accumulation in Rhodobacter sphaeroides.

Leucine deprivation of either phototrophically or chemotrophically growing cells of Rhodobacter sphaeroides resulted in a restriction in the continued accumulations of cellular RNA, phospholipids, and protein. Phototrophically growing cells also displayed restrictions in the accumulations of cellular carotenoids and bacteriochlorophyll. Leucine deprivation, however, did not provoke the accumulation of cellular ppGpp or alter the steady-state levels of ppGpp, ATP, or GTP in cells of R. sphaeroides.     

Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.     

Genetic and Non-Genetic Determinants of Raltegravir Penetration into Cerebrospinal Fluid: A Single Arm Pharmacokinetic Study

Background

Antiretroviral drugs vary in their central nervous system penetration, with better penetration possibly conferring neurocognitive benefit during human immunodeficiency virus (HIV) therapy. The efflux transporter gene ABCB1 is expressed in the blood-brain barrier, and an ABCB1 variant (3435C→T) has been reported to affect ABCB1 expression. The integrase inhibitor raltegravir is a substrate for ABCB1. We examined whether ABCB1 3435C→T affects raltegravir disposition into cerebrospinal fluid (CSF), and explored associations with polymorphisms in other membrane transporter genes expressed in the blood-brain barrier.

Methods

Forty healthy, HIV-negative adults of European descent (20 homozygous for ABCB1 3435 C/C, 20 homozygous for 3435 T/T, each group divided equally between males and females) were given raltegravir 400 mg twice daily for 7 days. With the final dose, plasma was collected for pharmacokinetic analysis at 9 timepoints over 12 hours, and CSF collected 4 hours post dose.

Results

The 4-hour CSF concentration correlated more strongly with 2-hour (r²=0.76, P=1.12x10^-11) than 4-hour (r²=0.47, P=6.89x10^-6) single timepoint plasma concentration, and correlated strongly with partial plasma area-under-the-curve values (AUC_0-4h r²=0.86, P=5.15x10^-16). There was no significant association between ABCB1 3435C→T and ratios of CSF-to-plasma AUC or concentration (p>0.05 for each comparison). In exploratory analyses, CSF-to-plasma ratios were not associated with 276 polymorphisms across 16 membrane transporter genes.

Conclusions

Among HIV-negative adults, CSF raltegravir concentrations do not differ by ABCB1 3435C→T genotype but strongly correlate with plasma exposure.

Trial Registration

ClinicalTrials.gov NCT00729924 http://clinicaltrials.gov/show/NCT00729924