Metabotropic Glutamate Receptors Inhibiting Excitatory Synapses in the CA1 Area of Rat Hippocampus

In the CA1 region of hippocampal slices prepared from young adult rats, we studied the ability of several specific agonists of metabotropic glutamate receptors (mGluRs) to depress excitatory synaptic transmission at the CA3–CA1 pyramidal cell synapses. Three groups of mGluRs have been described: group 1 (mGluR1 and 5) receptors are positively coupled to phospholipase C whereas group 2 (mGluR2 and 3) and group 3 (mGluR4, 6, 7 and 8) receptors are negatively coupled to adenylate cyclase. We found that the broad-spectrum agonist (1 S ,3R)-1-aminocyclopentyl-1,3-dicarboxylate and the group 1-specific agonist ( R,S )-dihydroxyphenylglycine both reversibly inhibited evoked field excitatory postsynaptic potentials, indicating the involvement of group 1 mGluRs. ( R,S )-3,5-dihydroxyphenylglycine presumably inhibited transmission via a presynaptic mechanism, as whole-cell voltage-clamp recordings revealed that inhibition of the synaptic transmission was always accompanied with an increase in paired-pulse facilitation. Treatment with a specific blocker of mGluR1 receptors, the phenylglycine derivative ( S )-4-carboxyphenylglycine, was without effect on the (1 S ,3 R )-1-amino-cyclopentyl-1,3-dicarboxylate-induced depression of the field excitatory postsynaptic potentials, strongly suggesting that mGluR5 receptors are responsible for the (1 S ,3 R )-1-aminocyclopentyl-1,3-dicarboxylate effect. Two selective agonists of group 2 mGluRs, (2 S ,1' s ,2' s )-2-(2'-carboxycyclopropyl)glycine and 4-carboxy-3-hydroxyphenylglycine, were totally ineffective in blocking CA3-CA1-evoked synaptic transmission, excluding the involvement of mGluR2/3 subtypes at this developmental stage.     

Aneurysm of the abdominal aorta in an eighteen-month-old child

We report the case of an infected aneurysm of the abdominal aorta in a 18 month-old child, discovered by routine palpation of the abdomen during hospitalization for pneumonia. Ultrasonography and arteriography showed a 6 cm aneurysm of the abdominal aorta beginning distal to the renal arteries which occluded the right common Iliac artery. The aneurysm was treated by interposing a 6 mm Gore-Tex graft between the infrarenal aorta and the aortic bifurcation. Pathologic examination of the aneurysmal wall demonstrated a leukocytic Infiltrate and the presence of encapsulated Gram positive organisms. Arterial aneurysms are exceedingly rare in children. Their etiology is varied: infection, connective tissue disease, trauma, inflammatory arterial disease or other rare diseases such as tuberous sclerosis, neurofibromatosis, or Beçhet’s disease.     

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.     

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.     

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.     

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH₂ and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min–hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH₂) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.     

Autoantibodies directed against the amino-terminal domain I of human calpastatin (ACAST-DI Ab) in connective tissue diseases. High levels of ACAST-DI Ab are associated with vasculitis in lupus

To identify new autoantibody populations in patients with rheumatic diseases, a cDNA expression library was immunoscreened with a rheumatoid arthritis (RA) patient's serum which contains autoantibodies binding to uncharacterized polypeptides by Western-blotting. One clone encoding the amino-terminal region (Nt) [domain L and half of domain I] of human calpastatin was selected. Different fragments of the selected cDNA were prepared and the corresponding recombinant polypeptides were produced by in vitro translation and analysed by Western blotting. Most RA sera bound to recombinant amino-terminal region and domain I but not to domain L. This prompted us to use a recombinant polypeptide corresponding to the domain I of calpastatin as the antigen in a solid-phase ELISA to test sera from patients with various systemic rheumatic diseases and healthy controls.Anti-calpastatin domain I antibodies (ACAST-DI Ab), were detected by ELISA in RA, systemic lupus erythematosus (SLE), Sj?gren's syndrome and control sera at respective frequencies of 10, 9, 0 and 1%. These Ab did not have prognostic value in early RA; high levels were significantly associated with vasculitis in SLE. Antibodies reacting with the calpastatin amino-terminal region are produced during systemic rheumatic diseases and are predominantly directed against domain I. High levels of these Ab may constitute a marker of vasculitis in SLE.     

Synaptic connectivity of serotonin graft efferents in the suprachiasmatic and supraoptic nuclei of the hypothalamus

We have previously reported that a cell suspension from the rostral part of the embryonic raphe grafted to the basal hypothalamus of 5,7-dihydroxytryptamine-denervated rats produced incomplete serotonin (5-HT) re-innervation of the suprachiasmatic nucleus (SCN) as opposed to hyper-innervation of the supraoptic nucleus (SON). We took advantage of this experimental model to investigate whether the graft-derived, 5-HT fibres retained normal ultrastructural features, and, particularly, a normal density of synaptic junctions, irrespective of the extent of target re-innervation. The intrinsic features of immunostained, graft-derived 5-HT axonal varicosities in both the SCN (ventral portion) and the SON were essentially similar to those exhibited by the respective endogenous innervation. Analysis of well-preserved varicosities in uninterrupted series of thin sections allowed us to evaluate directly the proportions of junctional to non-junctional 5-HT varicosities in both regions. Synaptic incidences were also remarkably conserved after grafting (45.5% in the SCN versus 38.5% in the SON; 48% and 38% in normal rats, respectively). Synapses were primarily reestablished on dendritic shafts, which also were identified as the major post-synaptic targets of the normal 5-HT innervations. We noted, however, a tendency toward increased numbers of symmetrical versus asymmetrical synapses in both the SCN and SON of grafted rats. Thus, irrespective of whether hypo-or hyper-innervation patterns developed post-grafting, the transplanted 5-HT neurons essentially retained normal ultrastructural features in their target territories, with a normal incidence of synaptic junctions. The data provide further support to the hypothesis that the innervation territory is the major determinant of the frequency with which ingrowing 5-HT fibres make synaptic junctions.