María Luisa Gómez-Lus

Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress.

BackgroundThe innate immune response is the first line of defence against invading microorganisms and it is also activated in different neurologic/neurodegenerative pathological scenarios. As a result, the family of the innate immune toll-like receptors (TLRs) and, in particular, the genetic/pharmacological manipulation of the TLR-4 signalling pathway emerges as a potential therapeutic strategy. Growing evidence relates stress exposure with altered immune responses, but the precise role of TLR-4 remains partly unknown.MethodsThe present study aimed to elucidate whether the elements of the TLR-4 signalling pathway are activated after acute stress exposure in rat brain frontal cortex and its role in the regulation of the stress-induced neuroinflammatory response, by means of its pharmacological modulation with the intravenous administration of the TLR-4 specific inhibitor TAK-242. Considering that TLR-4 responds predominantly to lipopolysaccharide from gram-negative bacteria, we checked whether increased intestinal permeability and a resultant bacterial translocation is a potential regulatory mechanism of stress-induced TLR-4 activation.ResultsAcute restraint stress exposure upregulates TLR-4 expression both at the mRNA and protein level. Stress-induced TLR-4 upregulation is prevented by the protocol of antibiotic intestinal decontamination made to reduce indigenous gastrointestinal microflora, suggesting a role for bacterial translocation on TLR-4 signalling pathway activation. TAK-242 pre-stress administration prevents the accumulation of potentially deleterious inflammatory and oxidative/nitrosative mediators in the brain frontal cortex of rats.ConclusionsThe use of TAK-242 or other TLR-4 signalling pathway inhibitory compounds could be considered as a potential therapeutic adjuvant strategy to constrain the inflammatory process taking place after stress exposure and in stress-related neuropsychiatric diseases.

Colonic bacterial translocation as a possible factor in stress-worsening experimental stroke outcome

Stress is known to be one of the risk factors of stroke, but only a few experimental studies have examined the possible mechanisms by which prior stress may affect stroke outcome. In stroke patients, infections impede neurological recovery and increase morbidity as well as mortality. We previously reported that stress induces a bacterial translocation and that prior immobilization stress worsens experimental stroke outcome through mechanisms that involve inflammatory mediators such as release of proinflammatory cytokines and enzyme activation. We now investigate whether bacterial translocation from the intestinal flora of rats with stress before experimental ischemia is involved in stroke outcome. We used an experimental paradigm consisting of exposure of Fischer rats to repeated immobilization sessions before permanent middle cerebral artery occlusion (MCAO). The presence of bacteria and the levels and expression of different mediators involved in the bacterial translocation were analyzed. Our results indicate that stress before stroke is related to the presence of bacteria in different organs (mesenteric nodes, spleen, liver, and lung) after MCAO and increases inflammatory colonic parameters (such as cyclooxygenase-2, inducible nitric oxide synthase, and myeloperoxidase), but decreases colonic immunoglobulin A, and these results are correlated with colonic inflammation and bacterial translocation. Understanding the implication of bacterial translocation during stress-induced stroke worsening is of great potential clinical relevance, given the high incidence of infections after severe stroke and their main role in mortality and morbidity in stroke patients.

Bacterial translocation affects intracellular neuroinflammatory pathways in a depression-like model in rats

Recent studies have suggested that depression is accompanied by an increased intestinal permeability which would be related to the inflammatory pathophysiology of the disease. This study aimed to evaluate whether experimental depression presents with bacterial translocation that in turn can lead to the TLR-4 in the brain affecting the mitogen-activated protein kinases (MAPK) and antioxidant pathways. Male Wistar rats were exposed to chronic mild stress (CMS) and the intestinal integrity, presence of bacteria in tissues and plasma lipopolysaccharide levels were analyzed. We also studied the expression in the prefrontal cortex of activated forms of MAPK and some of their activation controllers and the effects of CMS on the antioxidant Nrf2 pathway. Our results indicate that after exposure to a CMS protocol there is increased intestinal permeability and bacterial translocation. CMS also increases the expression of the activated form of the MAPK p38 while decreasing the expression of the antioxidant transcription factor Nrf2. The actions of antibiotic administration to prevent bacterial translocation on elements of the MAPK and Nrf2 pathways indicate that the translocated bacteria are playing a role in these effects. In effect, our results propose a role of the translocated bacteria in the pathophysiology of depression through the p38 MAPK pathway which could aggravate the neuroinflammation and the oxidative/nitrosative damage present in this pathology. Moreover, our results reveal that the antioxidant factor Nrf2 and its activators may be involved in the consequences of the CMS on the brain.

Morphological plasticity of Streptococcus oralis isolates for biofilm production, invasiveness, and architectural patterns

OBJECTIVE Streptococcus oralis is an early coloniser of the oral cavity that contributes to dental plaque formation. Many different genotypes can coexist in the same individual and cause opportunistic infections such as bacterial endocarditis. However, little is known about virulence factors involved in those processes. The aim was to analyze the evolving growth of S. oralis colony/biofilm to find out potentially pathogenic features. DESIGN Thirty-three S. oralis isolates were analyzed for: (1) biofilm production, by spectrophotometric microtiter plate assay; (2) colonial internal architecture, by histological methods and light and electron microscopy; (3) agar invasion, by a new colony-biofilm assay. RESULTS S. oralis colonies showed two different growth patterns: (1) fast growth rate without invasion or minimally invasive; (2) slow growth rate, but high invasion ability. 12.1% of strains were biofilm non-producers and 24.2% not invasive, compared to 51.5% biofilm high-producers and 39.4% very invasive. Both phenotypic characteristics tended to be mutually exclusive. However, a limited number of strains (15%) co-expressed these features at the highest level. CONCLUSIONS Morphological plasticity of S. oralis highlighted in this study may have important ecological and clinical implications. Coexistence of strains with different growth patterns could produce a synergic effect in the formation and development of subgingival dental plaque. Moreover, invasiveness might regulate dissemination and colonisation mechanisms. Simultaneous co-expression of high-invasive and high-biofilm phenotypes gives a fitness advantage during colonisation and may confer higher pathogenic potential.

Ex vivo bactericidal activity against group C Neisseria meningitidis in seronegative subjects.

SummaryBackground: To determine the anti-meningococcal C immunological activity by adding functional tests (opsonophagocytosis) to the classical serology techniques. Subjects and Methods: 42 adult volunteers were screened using serological methods (determination of total and bactericidal antibodies). Seronegative subjects were tested by opsonophagocytosis. Results: 24 subjects (57%) showed serological evidence of previous contact with Neusseria meningitidis group C antigens: 19 subjects had both total and bactericidal antibodies, two subjects had only total antibodies and three subjects had only bactericidal antibodies. Of the 18 seronegative subjects, five showed ex vivo activity in killing curves with or without polymorphonuclear cells: two subjects exhibited only complement-mediated bactericidal activity, one subject only opsonophagocytosis, and two subjects exhibited both activities. Conclusion: The addition of functional tests to the classical serological determination increases the evidence of previous contact with N. meningitidis antigens by 12%.

Qualitative and quantitative agar invasion test based on bacterial colony/biofilm.

Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms. This new methodology is simple, fast, reproducible, objective, inexpensive and can be used to study a great number of specimens simultaneously, all of which make it suitable for incorporation to the routine of any microbiology laboratory. It could also be a useful tool for additional studies related to clinical aspects of bacterial isolates such as virulence and antimicrobial response.

Efficacy of trovafloxacin in an in vitro pharmacodynamic simulation of an intraabdominal infection

An in vitro model simulating trovafloxacin concentrations in human serum after standard doses was used to investigate the activity of this drug with time against Bacteroides fragilis, Escherichia coli, Enterococcus faecalis and Staphylococcus aureus. Antibiotic concentrations used for each incubation period were: 4.24 mg/l (0-1 h), 3.69 mg/l (1-3 h), 3.25 mg/l (3-6 h), 2.38 mg/l (6-8 h), 1.35 mg/l (8-24 h). A 99.9% initial inoculum reduction (> 3 log10 cfu/ml) was defined as bactericidal activity. Bactericidal activity against these organisms was obtained with trovafloxacin after the first hour of incubation, and similar activity was obtained against B. fragilis, E. faecalis and S. aureus after 3 h, when they were tested individually. When the strains were tested as mixed culture, there was bactericidal activity against E. coli after 1 h incubation and after 3 h for S. aureus. This activity was observed against B. fragilis and E. faecalis after 6 h incubation in the mixed culture assays and after 3 h when organisms were tested individually. Regrowth was not observed over a 24 h period. These data show that trovafloxacin might be effective in intraabdominal infections caused by mixed aerobic and anaerobic microorganisms.