Regulation and restoration of motoneuronal synaptic transmission during neuromuscular regeneration in the pulmonate snail Helisoma trivolvis.

Regeneration of motor systems involves reestablishment of central control networks, reinnervation of muscle targets by motoneurons, and reconnection of neuromodulatory circuits. Still, how these processes are integrated as motor function is restored during regeneration remains ill defined. Here, we examined the mechanisms underlying motoneuronal regeneration of neuromuscular synapses related to feeding movements in the pulmonate snail Helisoma trivolvis. Neurons B19 and B110, although activated during different phases of the feeding pattern, innervate similar sets of muscles. However, the percentage of muscle fibers innervated, the efficacy of excitatory junction potentials, and the strength of muscle contractions were different for each cell's specific connections. After peripheral nerve crush, a sequence of transient electrical and chemical connections formed centrally within the buccal ganglia. Neuromuscular synapse regeneration involved a three-phase process: the emergence of spontaneous synaptic transmission (P1), the acquisition of evoked potentials of weak efficacy (P2), and the establishment of functional reinnervation (P3). Differential synaptic efficacy at muscle contacts was recapitulated in cell culture. Differences in motoneuronal presynaptic properties (i.e., quantal content) were the basis of disparate neuromuscular synapse function, suggesting a role for retrograde target influences. We propose a homeostatic model of molluscan motor system regeneration. This model has three restoration events: (1) transient central synaptogenesis during axonal outgrowth, (2) intermotoneuronal inhibitory synaptogenesis during initial neuromuscular synapse formation, and (3) target-dependent regulation of neuromuscular junction formation.

The PREVASC study: the clinical effect of a multifaceted educational intervention to prevent childhood asthma.

As asthma is the most common chronic disease in childhood, much attention is directed towards primary prevention. Here, the clinical effectiveness of a multifaceted educational prevention was studied. A total of 476 high-risk children were recruited during the prenatal period by general practitioners and randomised to either: 1) a control group, receiving usual care; or 2) an intervention group in which families received instruction from nurses on how to reduce exposure of newborns to mite, pet and food allergens, and passive smoking. A total of 443 infants were followed-up for 2 yrs. At 2 yrs of age, the intervention group (n = 222) had less asthma-like symptoms, including wheezing, shortness of breath and night-time cough, than the control group (n = 221). No significant differences in total and specific immunoglobulin E were found between the groups. During the first 2 yrs of life, the incidence of asthma-like symptoms was similar in both groups; however, subanalysis revealed a significant reduction in the female, but not in the male, intervention group. In conclusion, the intervention used in this study was not effective in reducing asthma-like symptoms in high-risk children during the first 2 yrs of life, although it was modestly effective at 2 yrs. Follow-up is necessary to confirm whether the intervention can actually prevent the development of asthma.

Compliance of asthmatic families with a primary prevention programme of asthma and effectiveness of measures to reduce inhalant allergens--a randomized trial.

BACKGROUND: Compliance to and the effect of pre- and post-natal exposure reduction measures to prevent asthma in high-risk children from asthmatic families were studied. METHOD: Families were randomized to a special care group (n=222) and a control group (n=221). Educational advice on measures to reduce their newborn's exposure to allergens and smoke was provided to the special care group during three visits (two pre-natal and one post-natal). The control group (n=221) received usual care. RESULT: After the intervention, the special care group differed significantly (P<0.01) from the usual care group in: use of anti-mite encasings (parental: 88% vs. 14%; baby: 98% vs. 10%); keeping pets outside (51% vs. 19%); combined breast- and hypoallergenic formula feeding (55% vs. 22%); first solids postponement until after the sixth month (71% vs. 28%); maternal post-natal smoking (52% vs. 28%). Little or no compliance was found for other sanitary measures (cleaning habits, providing a smooth floor covering, ventilation/airing, pet removal), exclusive breastfeeding, pre-natal smoking and partner smoking. In spite of pre-existent low allergen levels in both groups, there was a significant reduction of mite, cat, and dog allergens on the mattresses and mite and cat allergens in the living room in the special care group and were significantly lower compared with the usual care group after 1 year. CONCLUSION: High compliance was found for the use of anti-mite encasings; substantial compliance for using hypoallergenic formula, solid food postponement, keeping pets outside and reported post-natal maternal smoking. There was no compliance for sanitary measures and the reduction of maternal pre-natal passive smoking. Mite and pet allergens on mattresses were strongly reduced by anti-mite encasings.

Distribution of house dust mite allergen: comparing house dust mite allergen levels in dust samples collected from different sites on living room floors with smooth coverings.

BACKGROUND: The distribution of house dust mite allergen (Der p1) in living rooms with smooth floor coverings, as measured in the middle compared with the border of the floor was investigated. It was hypothesized that activity causes displacement of Der p1, from the middle towards the border. METHODS: Dust samples from the middle and border of 50 floors with smooth coverings were collected and analysed on Der p1 content in a standardized way. RESULTS: The Der p1 exposure expressed as per unit area (ng/m2) showed that border samples contained significantly more Der p1 compared with middle samples (median: 2.57 vs 0.27, respectively, P = 0.023). Presence of pets and presence of more than two inhabitants increased the difference. When expressed as per unit weight of dust (ng/g), significant differences were only detected when comparing Der p1 content of samples collected in households with three or more inhabitants [median: 2 (border) vs 53 (middle), respectively; P = 0.035]. CONCLUSIONS: The Der p1 is unequally distributed on living room floors with smooth coverings, most likely because of displacement of dust from the middle towards the border due to activity. Expression as ng/g of dust and ng/m2 could not obviously be interchangeable.

Phosphorylation-mimicking glutamate clusters in the proline-rich region are sufficient to simulate the functional deficiencies of hyperphosphorylated tau protein.

The microtubule-associated tau proteins represent a family of closely related phosphoproteins that become enriched in the axons during brain development. In Alzheimer's disease (AD), tau aggregates somatodendritically in paired helical filaments in a hyperphosphorylated form. Most of the sites that are phosphorylated to a high extent in paired helical filament tau are clustered in the proline-rich region (P-region; residues 172--251) and the C-terminal tail region (C-region; residues 368--441) that flank tau's microtubule-binding repeats. This might point to a role of a region-specific phosphorylation cluster for the pathogenesis of AD. To determine the functional consequences of such modifications, mutated tau proteins were produced in which a P- or C-region-specific phosphorylation cluster was simulated by replacement of serine/threonine residues with glutamate. We show that a phosphorylation-mimicking glutamate cluster in the P-region is sufficient to block microtubule assembly and to inhibit tau's interaction with the dominant brain phosphatase protein phosphatase 2A isoform AB alpha C. P-region-specific mutations also decrease tau aggregation into filaments and decrease tau's process-inducing activity in a cellular transfection model. In contrast, a phosphorylation-mimicking glutamate cluster in the C-region is neutral with regard to these activities. A glutamate cluster in both the P- and C-regions induces the formation of SDS-resistant conformational domains in tau and suppresses tau's interaction with the neural membrane cortex. The results indicate that modifications in the proline-rich region are sufficient to induce the functional deficiencies of tau that have been observed in AD. They suggest that phosphorylation of the proline-rich region has a crucial role in mediating tau-related changes during disease.

Interaction of tau with the neural membrane cortex is regulated by phosphorylation at sites that are modified in paired helical filaments.

The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995) J. Cell Biol. 131, 1327-1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser(199)/Ser(202), Thr(231), and Ser(396)/Ser(404)). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD.

Herpes simplex virus-mediated expression of the axonal protein tau in human model neurons (NT2-N cells).

The establishment of axonal-somatodendritic polarity is an important event during neuronal development. The analysis of the underlying molecular events requires experimental models that display characteristic steps in the development of polarity and that are accessible for experimental manipulations. Here we show that human model neurons (NT2-N cells) can be efficiently infected with an amplicon-based herpes simplex virus (HSV) system that expresses the axonal microtubule-associated protein tau. We demonstrate that the neurons express a high level of exogenous tau, which persists for several days, thus allowing us to analyze the morphological effects of the expressed protein. The intracellular interactions of tau and the effects on the microtubule structure of infected neurons, which were processed for immunocytochemistry, were determined using laser scanning microscopy (LSM). Exogenous tau expression does not result in an increased axon growth of the neurons but promotes neuronal microtubule assembly as indicated by an increased amount of total microtubule polymer as well as a labile, detyrosinated microtubule subpopulation. In contrast, tau expression does not induce a significant microtubule stabilization as judged from the quantitation of acetylated microtubule staining 24 hours after infection. The data demonstrate that HSV-mediated expression of proteins in human model neurons provides a useful system for analysis of the effect of neuronal proteins on the morphology and cytoskeletal organization of terminally differentiated polar neurons. In addition, it suggests a role for tau as a factor which locally promotes tubulin polymerization while the dynamics of axonal microtubules are preserved.

Immunocytological detection of isolated tumour cells in the bone marrow of malignant melanoma patients: a new method for the detection of minimal residual disease.

AIMS: Immunocytologically detected isolated tumour cells indicate a poor prognosis. This has been shown in breast, gastrointestinal and lung cancer, and might thereby help to indicate adjuvant therapy. Immunocytology has been proved to be a reliable technique and enables a phenotypic tumour cell characterization. We find this technique superior to molecular biological techniques such as reverse transcriptase polymerase chain reaction RT-PCR. So far, immunocytological studies have not been performed in malignant melanoma patients and our study aimed to establish this approach in melanoma patients. METHODS: Twenty melanoma patients who underwent surgery for lymph-node metastasis using a radical lymphadenectomy were studied. Using the immunoperoxidase method, cytospins of bone marrow aspirates (1.5x10(6)cells per patient) were stained with the monoclonal antibody HMB-45. Nineteen patients who were surgically treated but did not suffer from malignant melanoma were included as a control group. RESULTS: Four of the 20 patients showed isolated tumour cells in the aspirate. Three of these patients had stage IV disease. One patient had a stage III tumour (1/7; 14.3%). One patient was classified as stage II and did not show tumour cells in the bone marrow. No staining cells were found in the control group (n=19). CONCLUSIONS: Our study demonstrates that the immunocytological approach can be used as a new technique to detect occult tumour cell dissemination in malignant melanoma patients and supports previous findings in carcinoma of the stomach, colon, pancreas and other tumours.