Role of heparan sulphate proteoglycans as potential receptors for non-piliated Pseudomonas aeruginosa adherence to non-polarised airway epithelial cells

Tight junctions seal polarised surface epithelial respiratory cells so as to prevent the passage of bacteria and toxins through the epithelial sheet. Disruption of tight junctions, which may occur during injury and repair processes of airway epithelium, favours potential bacterial interaction with receptors from cell basolateral membranes. Earlier studies reported that non-polarised and untight epithelial respiratory cells are highly susceptible to Pseudomonas aeruginosa adherence and internalisation. As heparan sulphate proteoglycans (HSP) from cell basolateral membranes in epithelial cells without tight junctions may become accessible to bacterial ligands, the present study investigated their role as potential receptors for non-piliate P. aeruginosa ligands. Treatment of cells with heparitinase I and II significantly reduced (51.2% and 51.7%, respectively) P. aeruginosa adherence to epithelial respiratory cells without tight junctions. The internalisation of bacteria was not affected by treatment with heparitinases. Treatment of the bacteria with heparin and heparan sulphate also significantly reduced their adherence to respiratory cells (34.3% and 43.7%, respectively). Treatment of cells with other enzymes (trypsin, lipase and chondroitinase ABC) or treatment of bacteria with chondroitin-4-sulphate did not modify the adherence to respiratory cells significantly. Both affinity chromatography and Western blotting assays showed the interaction of different P. aeruginosa outer-membrane proteins (OMPs) with heparin. Several bacterial strains showed differences in their profile of heparin-binding OMPs, but all exhibited low mol. wt (< 30 kDa) reactive proteins. Reactivity of whole bacterial cells with heparin was also observed by transmission electron microscopy. These results suggest that HSP are potential receptors for P. aeruginosa adherence to non-polarised and untight epithelial respiratory cells.     

Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo

Heat shock protein 60 (HSP60) from Chlamydia pneumoniae was described to trigger in vitro inflammatory and cytokine responses including TNF and IL-12p40. Although it can be found in atherosclerotic plaques of patients, the stimulatory potential of chlamydial and other HSP60 in vivo is unclear. We now report that chlamydial HSP60 fails to induce TNF expression in vivo, and significant serum levels of IL-12p40 are only found upon intraperitoneal injection of high doses of HSP60 or after intravenous application. Upon purification of chlamydial HSP60 with polymyxin B-agarose columns, its ability to induce TNF secretion in vitro is much reduced. However, purified chlamydial HSP60 causes increased serum levels of the CXC chemokines KC and MIP2 in vivo, as well as a strong accumulation of polymorphonuclear neutrophils (PMN) in the peritoneal cavity upon intraperitoneal challenge. With respect to PMN accumulation, chlamydial HSP60 is more potent than endotoxin or the CpG oligonucleotide 1668. The responses observed are completely abolished in Toll-like receptor (TLR)2/4-double-deficient mice, while single-deficient mice respond almost normally. Furthermore, KC induction and PMN accumulation are largely dependent on MyD88. In conclusion, HSP60 from C. pneumoniae triggers inflammatory responses in vivo that differ from responses induced by endotoxin or CpG oligonucleotides and are dependent on TLR2 and 4.     

Genomic allelotyping for distinction of recurrent and de novo hepatocellular carcinoma after orthotopic liver transplantation.

Distinction between recurrent and de novo hepatocellular carcinoma (HCC) after orthotopic liver transplantation (OLT) bears important clinical and therapeutic implications. Techniques for molecular profiling of clinically suspected de novo and recurrent HCC are required since the histological/clinical discrimination of donor vs. recipient tumor origin is difficult. Multiple PCR amplification of 16 highly polymorphic short tandem repeat (STR) DNA sequences (routinely used for paternity and forensic assays) was applied in two patients who developed a second HCC after OLT. In both patients the technique provided reliable evidence that the two second HCC were recurrences of the primary tumor. Multiple STR genetic allelotyping is an effective tool for clear-cut discrimination of donor/recipient origin of a second HCC after OLT. Its application could be of great therapeutic relevance for such OLT patients.     

Histopathological aspects of Dengue-2 virus infected mice tissues and complementary virus isolation

The difficulty in studying dengue virus (DENV) infection in humans and in developing a virus vaccine is the absence of a suitable animal model which develops the full spectra of the Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Despite the fact that viruses have been found in various animal tissues, we isolated DENV from tissues of adult BALB/c mice, inoculated with DENV serotype 2 (DENV-2) obtained from human serum. Viruses were ultrastructurally identified and immunolocalized by immunofluorescence techniques in C6/36 mosquito cell cultures, inoculated with tissues (liver, lung, kidney and cerebellum) macerate supernatant from mice, 48 h post-infection (p.i.). These organs, collected at the same stage of infection, were examined histologically. The histopathological analysis revealed focal alterations in all tissues examined. Liver contained focal ballooned hepatocytes, but without modifying the average diameter of the majority of hepatocytes. Sinusoidal lumen was significantly diminished at this stage but portal and centrolobular veins became congested. Lungs exhibited hemorrhagic foci in the alveolar space, vascular congestion and focal alveolitis. Cerebellar tissue showed rare foci of neuronal compactation (Purkinje cells) and perivascular oedema. In kidneys it was observed an increase in glomerular volume with augmented endocapillary and mesangial cellularity, with reactivity to anti-IgM in all glomeruli of infected mice. In conclusion, DENV-2 was found in all tissues examined early in the evolution of infection. Presence of viruses in tissues has mainly led to hemodynamic alterations with generalized vascular congestion and increased permeability, and mast cell recruitment in lungs. The latter could participate in the vascular modifications in tissues.     

On the mechanisms of genotoxicity and metabolism of quercetin

Quercetin has been the subject of numerous studies on its genetictoxicity and carcinogenicity. Despite its well-proven geneticdamaging activity for various genetic end-points (reverse mutations,induction of SOS functions, induction of sister chromatid exchanges,chromosomal aberrations and micronuclei), the mechanisms ofgenetic damage by quercetin remain, by and large, unknown. Thepresent study aims to further extend the observations on thepossible active oxygen species mediated DNA-damaging activityof quercetin and the role of cytochrome P450-dependent metabolismon the genotoxicity of quercetin. The results reported in thiswork show that querceitn can produce the OH radical, as assessedby deoxyribose degradation in the presence of Fe^3³/EDTA(ethylenediaminetetraacetic acid), and that it induces strandbreakage in isolated plasmidic DNA (pUC18). The data supportthe hypothesis that the production of OH is mediated by H₂O₂.The results with genetically engineered V79 cells expressingrat cytochromes 1A1, 1A2 and 2B1 failed to demonstrate metabolismof quercetin, as indicated by the fact that neither an enhancementnor a decrease in the genotoxicity of quercetin was observed.Results obtained on the pH dependence of the induction of chromosomalaberrations by quercetin in V79 cells show that, as the valueof the medium is increased to 8.0, there is a significant increasein the number of aberrant cells, as expected if oxygen radicalsare responsible for the formation of chromosomal aberrations. ³To whom correspondence should be addressed     

Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs

Calcitonin gene-related peptide (CGRP) has been immunohistochemically co-localized with substance P (SP) in capsaicin-sensitive, varicose axons supplying the skin, viscera and cardiovascular system of the guinea pig. After treatment with colchicine in vitro, 82% of SP neurons in the dorsal root ganglia contained CGRP-like immunoreactivity while 96% of CGRP neurons were immunoreactive for SP. Both CGRP- and SP-like immunoreactive material are transported peripherally and centrally from dorsal root ganglia. Thus, in tissues such as the gut where there are intrinsic nerves containing SP but lacking CGRP, CGRP-like immunoreactivity is a useful means of specifically labelling axons of most sensory neurons containing SP.     

Co-localization of neuropeptide Y, vasoactive intestinal polypeptide and dynorphin in non-noradrenergic axons of the guinea pig uterine artery

Two major populations of perivascular axons containing immunoreactivity to neuropeptide Y (NPY) have been revealed in the main uterine artery of the guinea pig by immunohistochemical procedures which allow the simultaneous visualization of two antigens. One population contained immunoreactivity to dopamine-beta-hydroxylase (D beta H) and was presumably noradrenergic. The other main population of axons with NPY-like immunoreactivity (NPY-LI) did not have D beta H-like immunoreactivity (D beta H-LI) and was presumably non-noradrenergic. These non-noradrenergic axons also contained immunoreactivity to vasoactive intestinal polypeptide (VIP) and dynorphin (DYN). Indeed, nearly all axons with VIP-LI also contained NPY-LI and DYN-like immunoreactivity (DYN-LI). NPY constricted the uterine artery perfused in vitro, whilst VIP dilated uterine arteries preconstricted with noradrenaline or NPY. Thus, we have evidence for the coexistence of a vasoconstrictor peptide and a vasodilator peptide in the same non-noradrenergic perivascular axons, which also contain an opioid peptide, dynorphin.     

African swine fever virus-induced DNA polymerase is resistant to aphidicolin.

African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by phosphonoacetic acid and cytosine arabinoside. In contrast to all other alpha-like DNA polymerases of DNA viruses, ASFV-specific DNA polymerase is resistant to aphidicolin. Concentrations of the drug as high as 160 microM had no effect on virus production or plaquing efficiency. The resistance of ASFV DNA polymerase to aphidicolin was confirmed by analyzing the effect of the drug on viral DNA synthesis. A moderate inhibition of viral DNA synthesis was observed when aphidicolin was added immediately after virus adsorption but normal synthesis occurred, with a peak at 10 hr p.i., when the drug was added at 2 or 4 hr p.i. This suggests that a very early phase of ASFV DNA replication is sensitive to aphidicolin and is probably catalyzed by a different enzyme. An in vitro assay of DNA polymerase activity was used to assay the sensitivity of the virus-specific DNA polymerase to inhibitors. In correspondence to the results observed in vivo, phosphonoacetic acid strongly inhibited the enzyme activity, whereas aphidicolin had no effect. Resistance to aphidicolin was independent of the concentration of dCTP used in the assay. Three independent ASFV mutants resistant to phosphonoacetic acid showed the same resistance to aphidicolin as wild type virus.     

Genetic polymorphism of cytomegalovirus strains responsible of congenital infections

OBJECTIVES: Congenital Cytomegalovirus (CMV) infection is the main cause of neurological handicap in young children. The objective for studying genetic polymorphism of strains responsible for congenital infection is to identify CMV strains or groups of strains which would be more frequent in this context and/or which would be responsible for more severe congenital infection. METHODS: In this paper, we report and analyze the literature concerning the genetic polymorphism of CMV strains responsible of congenital infection, in the genes coding for the envelop protein B and the non structural UL144 protein and in the CMV short tandem repeats. RESULTS AND CONCLUSION: All UL144 and gB genotypes can be vertically transmitted from mothers to fetuses, none of these studies has shown any link between the genotypes and the severity of congenital disease. Moreover, no link between short tandem repeats polymorphism and severity of congenital disease has been demonstrated. However, short tandem repeats analysis may be a powerful tool to study the epidemiology of CMV congenital infections.