Essential role for cyclic AMP and its receptor protein in Yersinia enterocolitica virulence

Insertion mutations were isolated in cya and crp of Yersinia enterocolitica, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP). The cya and crp mutants were affected for the production of proteins exported by the Ysc, Ysa, and flagellar type III secretion systems (TTSS). Protein production by each TTSS was restored when the respective mutation was complemented by a plasmid-encoded copy of the wild-type gene. Both cya and crp mutants exhibited reduced virulence for orally infected BALB/c mice in a 50% lethal dose analysis. Examination of bacterial survival in host tissues showed that cya and crp mutants colonized Peyer's patches and, to a lesser extent, mesenteric lymph nodes. However, the mutants did not appear to disseminate to the liver and spleen of infected mice. An initial examination of the effectiveness of Y. enterocolitica cya and crp mutants to stimulate protective immunity against subsequent challenge with virulent bacteria in mice was promising. The results indicate that the cAMP-CRP regulatory system is required for Y. enterocolitica virulence.     

Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n=18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with 'malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n=26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n=17) designated LCT; and cytologically malignant (n=9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n=13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n=4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.     

A novel dystrophin isoform is required for normal retinal electrophysiology

Dystrophin is present in the outer plexiform layer of the retinaand is required for normal retinal function as measured by electroretinography.We describe the identification of a novel isoform of dystrophln(Dp260) present in the mouse retina. The unIque 5' terminusof the mRNA originates from a newly identified exon and is splicedin frame to exon 30 of the Duchenne muscular dystrophy (DMD)gene. The retinal isoform of dystrophln has 13 novel amino acidsas its N-terminus followed by most of the dystrophin rod domainand the cysteine-rich C-terminal domains. Analysis of mousetissues indicated this isoform of dystrophin Is expressed inretina, brain and cardiac tissue. Comparison of retinal electrophysiologyin mdx and mdx^cv3 mouse suggests that Dp260 is required fornormal retinal function.     

Composite articular cartilage engineered on a chondrocyte-seeded aliphatic polyurethane sponge

To circumvent the reconstructive disadvantages inherent in resorbable polyglycolic acid (PGA)/polylactic acid (PLA) used in cartilage engineering, a nonresorbable, and nonreactive polyurethane sponge (Tecoflex sponge, TS) was studied as both a cell delivery device and as an internal support scaffolding. The in vitro viability and proliferation of porcine articular chondrocytes (PACs) in TS, and the in vivo generation of new articular cartilage and long-term resorption, were examined. The initial cell attachment rate was 40%, and cell density increased more than 5-fold after 12 days of culture in vitro. PAC-loaded TS blocks were implanted into nude mice, became opalescent, and resembled native cartilage at weeks 12 and 24 postimplantation. The mass and volume of newly formed cartilage were not significantly different at week 24 from samples harvested at week 6 or week 12. Safranin O-fast green staining revealed that the specimens from cell-loaded TS groups at week 12 and week 24 consisted of mature cartilage. Collagen typing revealed that type II collagen was present in all groups of tissue-engineered cartilage. In conclusion, the implantation of PAC-TS resulted in composite tissue-engineered articular cartilage with TS as an internal support. Long-term observation (24 weeks) of mass and volume showed no evidence of resorption.     

An enzyme-linked immunosorbent assay (ELISA) for the major crustacean allergen, tropomyosin, in food

Shellfish are a common cause of food reactions in hypersensitive individuals and are among the eight foods that account for over 90% of food allergies. At present, the only way to prevent these serious consequences of food allergies is to avoid the foods that trigger the reactions. A sandwich-ELISA kit has been developed for the detection of crustacean meat in food, based on the major heat-stable shellfish allergen, tropomyosin. Tropomyosin was purified from whole prawn (Penaeus latisulcatus) and used to immunize rabbits after confirming its identity by MALDI-TOF MS. A sandwich-ELISA based on the rabbit antibodies takes less than 2 h to perform, including the food extraction, and has a detection limit of 1 ppm crustacean (prawn, lobster), without detectable cross-reactivity with fish or mammalian meat.     

Vocalizations in the cat: behavioral methodology and spectrographic analysis

Summary Attempts to understand the neural mechanisms underlying mammalian vocal behaviors, including speech, require study of the neural activity and anatomy of vocalization-controlling brain structures. Such studies necessitate the application of invasive neurobiological techniques in animal models. In the current study, cats are used in the development of an animal model of vocal tract control. The animals are instrumentally conditioned to vocalize for food reward. Acquisition of this task can occur within a few minutes, although additional training generally is required to solidly establish the behavior and to train subjects to produce consistently high rates of vocalization for prolonged periods of time. Following training, animals can generally sustain a rate of two calls per minute for a period of over two hours. Optimal task performance is partly dependent on motivation level. Although there is considerable variation between animals, the vocalizations produced have an average duration of 600 ms and a fundamental frequency of around 500 Hz. In addition, during a typical vocalization, there are dynamic variations of about 150 Hz for fundamental frequency and 17 dB for sound intensity. These variations provide opportunities for relating neural and muscular activity to different aspects of the vocal behavior they control. Based on a number of considerations, the model and techniques discussed here probably are most applicable to studying the neurobiology of sub-cortical nuclei subserving vocal control. Similar mechanisms might well be present in other species, including humans. Thus, data obtained from study of this model may be applicable to understanding the processes underlying vocal tract control during human speech.     

Vascularization and tissue infiltration of a biodegradable polyurethane matrix

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 microm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications.