Superinfecting mycobacteria home to established tuberculous granulomas

A central paradox of tuberculosis immunity is that reinfection and bacterial persistence occur despite vigorous host immune responses concentrated in granulomas, which are organized structures that form in response to infection. Prevailing models attribute reinfection and persistence to bacterial avoidance of host immunity via establishment of infection outside primary granulomas. Alternatively, persistence is attributed to a gradual bacterial adaptation to evolving host immune responses. We show here that superinfecting Mycobacterium marinum traffic rapidly into preexisting granulomas, including their caseous (necrotic) centers, through specific mycobacterium-directed and host cell-mediated processes, yet adapt quickly to persist long term therein. These findings demonstrate a failure of established granulomas, concentrated foci of activated macrophages and antigen-specific immune effector cells, to eradicate newly deposited mycobacteria not previously exposed to host responses.     

Chronic ethanol exposure differentially regulates NOS1 mRNA levels depending on rat brain area

In vitro experiments were performed on brainstem – spinal cord preparations from mouse neonates to compare the noradrenergic regulations of the respiratory network in the control C3H/HeJ strain and the transgenic Tg8 strain which has been created from the C3H/HeJ strain by deletion of the gene encoding monoamine oxidase A (MAOA), the main enzyme for serotonin degradation. In both control and MAOA-deficient strains, we show: (i) that the pontine A5 area exerts a potent inhibitory modulation on the respiratory rhythm generator; (ii) that noradrenaline application induces a tonic phrenic activity; and (iii) that noradrenaline increases the respiratory rhythm. The latter effect is however delayed and weak in the Tg8 strain. Therefore, MAOA-deficiency has only slightly altered the noradrenergic regulations of the respiratory network.     

Antifungal coating by biofunctionalized polyelectrolyte multilayered films

The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. Here, we experiment a new type of topical antifungal coating using the layer-by-layer technique. The nanometric multilayer film obtained by this technique is functionalized by the insertion of a chromogranin A-derived antifungal peptide (CGA 47-66, chromofungin). We show that the embedded peptide keeps its antifungal activity by interacting with the fungal membrane and penetrating into the cell. In vitro studies demonstrate that such an antifungal coating is able to inhibit the growth of yeast Candida albicans by 65% and completely stop the proliferation of filamentous fungus Neurospora crassa. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly(methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an in vivo oral candidiasis rat model. Taken together, these results assessed the functionalized multilayer films containing a new potent antifungal non-toxic peptide, as a novel and promising technique for local antifungal protection.     

Variety and complexity of chromosome 17 translocations in neuroblastoma

In neuroblastoma, the most frequent genetic alteration is gain of chromosome arm 17q, which arises from unbalanced translocations. To document these genetic events more precisely, we performed an extensive study of chromosome 17 breakpoints in 27 neuroblastoma cell lines by using a combination of fluorescence in situ hybridization mapping with BAC/PAC clones and allele analysis with polymorphic markers. All cases exhibited one or more unbalanced chromosome 17 translocations, and 15 distinct breakpoint regions could be mapped. This high variability indicates that gene fusion or disruption events are extremely unlikely to account for the underlying oncogenic role of these translocations. However, breakpoints were not randomly distributed, most of them mapping to the proximal part of 17q. As a result of translocations, all cell lines but one exhibited gain of the 53.5 Mb-->qter fragment, bordered proximally by the clone CTC-462L7. The most telomeric breakpoint, flanked by the clone RP11-443M10, defined the 70.9 Mb-->qter fragment as a region of additional gain. In addition to chromosome gains, loss of heterozygosity for the short arm of chromosome 17 was observed in close to half the cases. It was either related to a chromosome 17 monosomy or to a uniparental isodisomy. Finally, in cases with a single normal chromosome 17, we show that the parental origin of the translocated chromosome 17 can be either distinct or identical to that of the normal chromosome. Similarly, multiple translocations within the same cell line can either involve the same or different chromosome 17 homologues, indicating the likely absence of parental origin bias in the generation of these alterations.     

Structure of four amplified DNA novel joints

The structures of four novel joints present in the amplified DNA of a Syrian hamster cell line highly resistant to N-(phosphonacetyl)-l-aspartate were analyzed. Novel joints J1, J2, and J4 were formed by recombination between two regions of wild-type DNA, whereas joint J3 is the end point of an inverted duplication. A fraction of the J3 copies displays a cruciform structure in the purified genomic DNA. The formation of J1 and J2 apparently involved a simple breakage and joining of the two wild-type sequences, whereas extra nucleotides are present at the junction point of J3 and J4. The two regions of the wild-type DNA which have recombined to form J1, J2, and J4 show few sequence similarities, indicating that these joints probably resulted from nonhomologous recombination. AT-rich regions are present in the vicinity of the breakpoint for the four joints and eight of 10 crossover points could be associated with putative topoisomerase I cleavage sites. Our results indicate that different types of novel joints are present in the amplified DNA of this cell line, which was isolated after several steps of selection.     

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10⁻⁶M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.     

Study of the genotoxic potential of 17 mycotoxins with the SOS Chromotest

Seventeen mycotoxins [aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₂),G₂ (AFG₂), aflatoxicol, sterigmatocystin, patulin, citrinin,penicillic acid, T-2 toxin, diacetoxyscirpenol, zeara-lenone,zearalenol ( and ß isomers 1:1), ochratoxin A, norso-lorinicacid, averufin, versicolorin A] were tested using the SOS Chromotest(PQ37 and PQ35). Six of the mycotoxins (AFB₁, AFG₁, AFB₂, aflatoxicol,sterigmatocystin and versicolorin A) were genotoxic on PQ37strain with and without metabolic activation. The results obtainedwith metabolic activation are in agreement with positive resultsobtained in other tests of genotoxicity. Except for AFB₂, thepresence of a double bond C₈–C₉ in the dihydrobenzofurane(DHBF) ring explained the activity due to the formation of anepoxide, but the coumarin cyclopentenone ring also plays a rolein the qualitative differences of genotoxic activity. The wild-typeuvrB gene in PQ35 decreases the genotoxic response with andwithout metabolic activation. Without metabolic activation,only mycotoxins possessing the DHBF ring group and double linkageC₈–C₉ exhibit a genotoxic effect. ³To whom correspondence should be addressed     

Performance of the new VITEK 2 GP card for identification of medically relevant gram-positive cocci in a routine clinical laboratory

The VITEK 2 gram-positive (GP) identification card (bioMerieux, Marcy l'Etoile, France) has been redesigned to achieve greater accuracy in the identification of gram-positive cocci. A total of 43 biochemical tests, including 17 enzymatic tests, are present in the card and interpreted in a kinetic mode, for up to 8 h. The VITEK 2 database, used in conjunction with the GP identification card, allows the identification of 115 different taxa. A total of 364 strains of GP cocci (217 Streptococcaceae strains and 147 Micrococcaceae strains) belonging to 31 taxa were tested with the new VITEK 2 GP identification card. Of the 364 strains, 105 were taken from routine primary plating media. A total of 344 strains (94.5%) were correctly identified to the species level and 17 strains (4.7%) were identified with low discrimination, requiring additional tests, whereas 1 strain (0.3%) was incorrectly identified and 2 strains (0.5%) remained unidentified. Within 7 h of the start of incubation, more than 90% of all strains were identified. Of the 105 primary cultures, 97% were correctly identified to the species level, 2% were identified with low discrimination, and 1% remained unidentified. Identification performance data were independent of each of the three plating media used. It is concluded that the new VITEK 2 GP identification card provides reliable results for the identification of GP cocci under routine laboratory conditions.