Immunosuppressive but non-LasR-inducing analogues of the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (1) is involved not only in bacterial activation but also in subversion of the host immune system, and this compound might thus be used as a template to design immunosuppressive agents, provided derivatives devoid of quorum-sensing activity could be discovered. By use of a leukocyte proliferation assay and a newly developed bioluminescent P. aeruginosa reporter assay, systematic modification of 1 allowed us to delineate the bacterial LasR-induction and host immunosuppressive activities. The main determinant is replacement of the methylene group proximal to the β-ketoamide in the acyl chain of 1 with functions containing heteroatoms, especially an NH group. This modification can be combined with replacement of the homoserine lactone system in 1 with stable cyclic groups. For example, we found the simple compound N(1)-(5-chloro-2-hydroxyphenyl)-N(3)-octylmalonamide (25d) to be over twice as potent as 1 as an immune suppressor while displaying LasR-induction antagonist activity.     

The cell-specific phenotype of the polymorphic vacA midregion is independent of the appearance of the cell surface receptor protein tyrosine phosphatase beta

There are two alleles, m1 and m2, of the midregion of the vacuolating cytotoxin gene (vacA) of Helicobacter pylori which code for toxins with different cell specificities. Here we describe the construction of five chimeric strains in which regions of vacA were exchanged between the two genotypes. By analyzing the toxicity of these strains for HeLa and RK13 cells we have confirmed that a 148-amino-acid region determines the phenotypic differences between the two forms of the protein and that this entire region is important for cytotoxicity. Furthermore, we have used our chimeric strains to investigate whether variations in the midregion of VacA have an effect on phorbol 12-myristate 13-acetate (PMA)-induced VacA sensitivity in HL-60 cells. The PMA-induced VacA sensitivity of HL-60 cells has been previously associated with the appearance of the cell surface receptor protein tyrosine phosphatase beta (RPTPbeta). Our data indicate that both the m1 and m2 forms of VacA are able to utilize RPTPbeta, and the cell-specific phenotype of the midregion is independent of the presence of RPTPbeta. It appears that another as-yet-unidentified receptor exists in HL-60 cells that accounts for the m2 phenotype in this cell line. Also, by studying the effect of PMA on levels of RPTPbeta in other cell lines and toxicity of VacA in these cell lines we have shown that RPTPbeta does not play a major role in the vacuolation of HeLa cells.     

Body fluid derived exosomes as a novel template for clinical diagnostics

Background

GH and IGFs serum levels decline with age. Age-related changes appear to be associated to decreases in these anabolic hormones. We have previously demonstrated that IGF-I replacement therapy improves insulin resistance, lipid metabolism and reduces oxidative damage (in brain and liver) in aging rats. Using the same experimental model, the aim of this work was to study whether the exogenous administration of IGF-II, at low doses, acts analogous to IGF-I in aging rats.

Methods

Three experimental groups were included in this study: young healthy controls (yCO, 17 weeks old); untreated old rats (O, 103 weeks old); and aging rats treated with IGF-II (O+IGF-II, 2 μg * 100 g body weight^-1 * day^-1) for 30 days. Analytical parameters were determined in serum by routine laboratory methods using an autoanalyzer (Cobas Mira; Roche Diagnostic System, Basel, Switzerland). Serum levels of hormones (testosterone, IGF-I and insulin) were assessed by RIA. Serum Total Antioxidant Status was evaluated using a colorimetric assay. Mitochondrial membrane potential was evaluated using rhodamine 123 dye (adding different substrates to determine the different states). ATP synthesis in isolated mitochondria was determined by an enzymatic method.

Results

Compared with young controls, untreated old rats showed a reduction of IGF-I and testosterone levels with a decrease of serum total antioxidant status (TAS). IGF-II therapy improved serum antioxidant capability without modifying testosterone and IGF-I circulating concentrations. In addition, IGF-II treatment reduced oxidative damage in brain and liver, improving antioxidant enzyme activities and mitochondrial function. IGF-II was also able to reduce cholesterol and triglycerides levels increasing free fatty acids concentrations.

Conclusions

We demonstrate that low doses of IGF-II induce hepatoprotective, neuroprotective and metabolic effects, improving mitochondrial function, without affecting testosterone and IGF-I levels.     

Follow-up of newborns treated with extracorporeal membrane oxygenation: a nationwide evaluation at 5 years of age

Introduction  

Extracorporeal membrane oxygenation (ECMO) is a supportive cardiopulmonary bypass technique for babies with acute reversible cardiorespiratory failure. We assessed morbidity in ECMO survivors at the age of five years, when they start primary school and major decisions for their school careers must be made.     

Characterization of an immunoreactive protein from the agent of human granulocytic ehrlichiosis.

A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.