Laryngeal pleomorphic rhabdomyosarcoma

A case of an extremely unusual tumor of the larynx, pleomorphic rhabdomyosarcoma, is presented with a review of literature. This is the fifth case of this malignancy described in the larynx in the English language literature. A histopathological diagnosis was made with immunohistochemistry and electron microscopy. In contrast to other reported cases, the tumor in the present case had a very aggressive behavior. Despite radical surgery involving total laryngectomy and neck dissection followed by radiation therapy, the patient died of disease 8 months following treatment. Received: 14 March 1997 / Accepted: 23 January 1998     

A complementarity-determining region peptide of an anti-desmosome autoantibody may interact with the desmosomal plaque through molecular mimicry with a cytoplasmic desmoglein 1 sequence

The sera of patients with pemphigus, a group of autoimmune blistering skin diseases, contain autoantibodies directed against components of adhering junctions termed desmosomes. F12, a human monoclonal antibody derived from a pemphigus patient, recognizes an unknown polypeptide of the desmosomal and hemidesmosomal plaques. The third complementarity-determining region of the F12 heavy chain (VH-CDR3) was shown to share a four-amino-acid sequence (GSSG) with the intracellular domains of desmoglein 1 and bullous pemphigoid antigen 2 which interact with components of, respectively, the desmosomal and hemidesmosomal plaques. Computer modeling of F12 showed that the GSSG sequence protudes inside the antigen-combining site and thus might be involved in antigen interactions. The GSSG sequence is essential to F12 function, since a peptide containing the VH-CDR3 inhibited its binding to target antigens while VH-CDR3 peptides with specific modifications of the GSSG sequence did not. These data allow us to hypothesize that certain autoantibodies produced during the course of an autoimmune disease can behave as adhesion molecules, through the molecular mimicry of the motif involved in protein/protein adhesion, and to propose a new self-antigen binding mechanism for some autoantibodies.     

Kinetic and biologic properties of recombinant ChIFN-gamma expressed via CELO-virus vector.

In this study, a replicative fowl adenovirus serotype 1 (CELO) recombinant expressing chicken interferon-gamma (ChIFN-gamma) was constructed. In the engineered recombinant, the ChIFN-gamma gene was placed under the control of cytomegalovirus (CMV) promoter. The ChIFN-gamma expression cassette was inserted in the right end of the CELO genome (D fragment), which was able to carry the largest insertion of foreign DNA without affecting the replication functions of the vector. The recombinant ChIFN-gamma (rChIFN-gamma) produced in the CELO-virus expression system was characterized by comparing its biologic activities with that of rChIFN-gamma produced via the baculovirus expression system (Bac-ChIFN-gamma). CELO-ChIFN-gamma inhibited the replication of cytolytic virus in chicken embryo fibroblasts (CEFs) and activated macrophages in a better manner than did Bac-ChIFN-gamma . Moreover, the in vitro and in vivo stability of the CELO-derived rChIFN-gamma was considerably higher than that of the Bac-ChIFN-gamma. The CELO-ChIFN-gamma recombinant vector was able to replicate in vitro in the loghorn male hepatoma (LMH) hepatocyte cell line and to produce detectable levels of recombinant cytokine in supernatant as early as 90 min post-infection. Therefore, the CELO-virus expression system is an appropriate system for high-level expression of biologically active and stable ChIFN-gamma.     

A novel nucleotide-containing antigen for human blood γδ T lymphocytes

The stimulation of human γδ T cells by mycobacteria occurs through recognition of four distinct nonpeptide phosphorylated antigens termed TUBag1–4. Among these latter, TUBag4 has already been biochemically characterized as a γ-X derivative of 5′-deoxythymidine triphosphate (Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M. and Fournié, J.-J., Science 1994. 264: 267). However, despite chemical synthesis of weakly stimulatory nucleotide-containing analogs, these mycobacterial compounds remained the sole nucleotide-containing antigens actually isolated from natural sources. Here, we present the complete isolation of the TUBag3 antigen from Mycobacterium fortuitum and demonstrate that this nonpeptide molecule contains a 5′-UTP nucleotide moiety. On selected Vγ9/Vδ2 clones, T cell responses can be triggered with nanomolar concentrations of TUBag3. Like crude mycobacterial extracts, this purified nucleotide conjugate elicits a strong polyclonal response of γδ PBL from healthy donors. Furthermore, we present evidence that this compound is distinct from the recently synthesized γ-isopentenyl 5′-UTP, a nucleotide conjugate of isopentenyl pyrophosphate that was found to be stimulatory for human γδ T cells (Tanaka, Y., Morita, C. T., Tanaka, Y., Nieves, E., Brenner, M. B. and Bloom, B. R., Nature 1995. 375: 155). Since it appears that both mycobacterial nucleotide antigens are molecules structurally related to peculiar precursors of nucleic acid synthesis, we propose that TUBag-reactive T cells might be specifically devoted to surveillance of proliferating cells.     

Sequence-specific intercalating agents: intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide-intercalator conjugates.

An acridine derivative was covalently linked to the 5' end of a homopyrimidine oligonucleotide. Specific binding to a homopurine-homopyrimidine sequence of duplex DNA was demonstrated by spectroscopic studies (absorption and fluorescence) and by "footprinting" experiments with a copper phenanthroline chelate used as an artificial nuclease. A hypochromism and a red shift of the acridine absorption were observed. Triple-helix formation was also accompanied by a hypochromism in the ultraviolet range. The fluorescence of the acridine ring was quenched by a stacking interaction with a G.C base pair adjacent to the homopurine-homopyrimidine target sequence. The intercalating agent strongly stabilized the complex formed by the oligopyrimidine with its target duplex sequence. Cytosine methylation further increased the stability of the complexes. Footprinting studies revealed that the oligopyrimidine binds in a parallel orientation with respect to the homopurine-containing strand of the duplex. The intercalated acridine extended by 2 base pairs the region of the duplex protected by the oligopyrimidine against degradation by the nuclease activity of the copper phenanthroline chelate. Random intercalation of the acridine ring was lost due to the repulsive effect of the negatively charged oligonucleotide tail. Intercalation occurred only at those double-stranded sequences where the homopyrimidine oligonucleotide recognized the major groove of duplex DNA.     

Ontogenetic profile of glutamate uptake in brain structures slices from rats: sensitivity to guanosine

The excitotoxicity of the neurotransmitter glutamate has been shown to be connected with many acute and chronic diseases of the CNS. High affinity sodium-dependent glutamate transporters play a key role in maintaining adequate levels of extracellular glutamate. In the present study, we used slices of striatum, hippocampus and cortex from rat brain to describe the in vitro profile of glutamate uptake during development and ageing, and its sensitivity to guanosine. In all structures, glutamate uptake was higher in immature animals. There was a maximum decrease in glutamate uptake in striatum and hippocampus in 15-month-old rats, which later increased, while in cortex there was a significant decrease in rats aged 60 days old. The effect of guanosine seems to be age and structure dependent since the increase in basal glutamate uptake was only seen in slices of cortex from 10-day-old animals.     

Epidermal growth factor receptor regulates normal urothelial regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1). a specific tyrosine kinase inhibitor (AG1478) and (2). a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-alpha and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.     

Hemocompatibility of medical connectors with biopassive or bioactive surface coatings

Although medical connectors compose very small parts of the extracorporeal circulation (ECC) system they represent a critical localization where early thromboembolic processes can manifest. In the present study we modified an in vitro closed-loop model with fresh human whole blood for the preclinical evaluation of the hemocompatibility of three types of medical connectors: non-coated (control); with silicone-, and heparin-coating. Each single loop consists of five polycarbonate connectors joined together by five pieces of silicone tubes. Thrombin-antithrombin-III, beta-thromboglobulin (beta-TG), PMN-Elastase, terminal complement complex, CD 11b expression, and surface-absorbed fibrinogen were measured. After 1 and 2 h recirculation, platelet loss, release of beta-TG, and adsorption of fibrinogen were significantly higher (p<0.05) within the non-coated connectors compared to the silicone- and heparin-coated groups. Following this experiment, the connectors were filled again with fresh heparinized whole blood from the same donor to evaluate the influence of prior blood contact. Here, the activation of platelets and coagulation was dependent on the duration of the blood preincubation period. Probably, the coated surfaces possess a reduced, or selective adsorption of plasma proteins, which in turn leads to a faster creation of a blood-friendly secondary superficial membrane, and prevents a further denaturation and hence activation of the adsorbed proteins.