Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

OBJECTIVE AND DESIGN: Currently no method is available for measurement of mediator release from intact human lung. In this study, a microdialysis technique was used to measure histamine release from mast cells in human lung tissue ex vivo. MATERIAL: Microdialysis fibers of 216 microm were inserted into lung tissue and perfused with Krebs Ringer buffer at a rate of 3 microl/min. After a 15 min period of steady-state perfusion, anti-IgE and vehicle were injected into the lung tissue above individual fibers. Samples from each fibre were collected for 20 min at 2 min intervals. Histamine was assayed fluorometrically. RESULTS: Anti-IgE concentrations of 40-40,000 U/ml dose-dependently released histamine, significant histamine release being demonstrated with anti-IgE concentrations of 400 U/ml and greater. The kinetics of histamine release showed peak values 2-8 min after the injection. Great individual responses were observed but data could be reproduced within individual donors. Monocyte chemoattractant protein-1, a potent basophil secretagogue, did not induce histamine release in lung tissue which indicated mast cells to be the histamine source. Substance P did not release histamine in the lung tissue. CONCLUSIONS: The microdialysis technique allowed measurements of histamine release from mast cells in intact lung ex vivo. The method may prove useful since a number of experiments can be performed in a few hours in intact lung tissue without any dispersion or enzymatic treatment.     

Neuroleptic use and behavioral disturbance in nursing homes: a 1-year study

This article discusses a longitudinal study of change in disruptive behaviors among nursing home residents treated with neuroleptics compared with those not treated with neuroleptics. Observations were made of 201 participants on admission to and after 1 year in eight skilled nursing facilities. Nine disruptive behaviors were measured using the Psychogeriatric Dependency Rating Scale with nursing assistants. Neuroleptic use was documented from medication records. Odds ratios are reported for the association of behavior at baseline and use of neuroleptics on nine problem behaviors. For those who received neuroleptics during the year, there was greater change in both developing and resolving disruptive behaviors than for those not receiving neuroleptics. For both groups, restless or pacing behavior and belligerent behavior manifested by refusing instructions changed the most, both in developing and in apparently resolving. Our results show that change in disruptive behaviors occurs among nursing home residents regardless of neuroleptic use, but it occurs more frequently among those who receive neuroleptic medication. Knowledge of which disruptive behaviors are most likely to resolve or develop is important in training nursing home staff to cope with the behaviors as well as in planning interventions that may modify such behaviors.     

Crystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residues

BACKGROUND AND PURPOSE: Early and accurate diagnosis of brain edema in stroke patients is essential for the selection of appropriate treatment. We examined the correlations between the changes in the apparent diffusion coefficient (ADC), regional water content, and tissue ultrastructure during early focal cerebral ischemia. METHODS: The left middle cerebral arteries of cats were occluded with an intramagnet occlusion/recirculation device. T2-weighted, diffusion-weighted, and perfusion imaging were performed repeatedly during the initial 3 hours after occlusion. The ADCs obtained from ADC maps were compared with the corresponding tissue water content values determined by gravimetry and electron microscopic water localization. RESULTS: ADC reduction was detected in areas of low perfusion 15 minutes after occlusion and thereafter. The water content increase correlated linearly with the ADC decreases in both the gray and white matter. However, both the water content corresponding to an ADC value and the rate of ADC change of the gray and white matter differed significantly (P<.05) as follows: y = -10105x + 8533 (r=.86) and y = -6174x + 4611 (r=.67), respectively, where x is the water content (grams water per gram tissue) and y is the ADC (x 10(-6) mm2/s). Hydropic astrocytic swelling was seen in both structures, and in the white matter, oligodendroglial and myelinated axonal swelling and periaxonal space enlargement were observed. CONCLUSIONS: When early ischemic edema in experimental focal cerebral ischemia is evaluated with ADC mapping, the different slopes and intercepts of the water content and ADC correlation lines for the gray and white matter, which probably reflect different ultrastructural localization of water, should be taken into account.     

Cardiac dysrrhthmia in systemic disease

This is an ECG of a 6-year-old girl. She presented to us for management of epilepsy. Magnetic resonance imaging (MRI) of the brain showed multiple lesions consistent with cortical and subcortical tubers. There were also achromic spots on her skin and echocardiography demonstrated a rhabdomyoma near the right ventricular outflow tract. A diagnosis of tuberous sclerosis, an autosomal-dominantly inherited condition, was made. What is abnormal about the ECG?     

The Contribution of Small Vessel Disease to Neurodegeneration: Focus on Alzheimer’s Disease,Parkinson’s Disease and Multiple Sclerosis

Brain small vessel disease (SVD) refers to a variety of structural and functional changes affecting small arteries and micro vessels, and manifesting as white matter changes, microbleeds and lacunar infarcts. Growing evidence indicates that SVD might play a significant role in the neurobiology of central nervous system (CNS) neurodegenerative disorders, namely Alzheimer’s disease (AD) and Parkinson’s disease (PD), and neuroinflammatory diseases, such as multiple sclerosis (MS). These disorders share different pathophysiological pathways and molecular mechanisms (i.e., protein misfolding, derangement of cellular clearance systems, mitochondrial impairment and immune system activation) having neurodegeneration as biological outcome. In these diseases, the actual contribution of SVD to the clinical picture, and its impact on response to pharmacological treatments, is not known yet. Due to the high frequency of SVD in adult-aged patients, it is important to address this issue. In this review, we report preclinical and clinical data on the impact of SVD in AD, PD and MS, with the main aim of clarifying the predictability of SVD on clinical manifestations and treatment response.     

Staphylococcal enterotoxin B primes cytokine secretion and lytic activity in response to native bacterial antigens

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-gamma. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to "bystander" fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-gamma decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.     

Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (approximately 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca2+]free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca2+-triggering step, blocks complex disruption by Ca2+. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca2+-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion-clamp."     

Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identified Drosophila neuromuscular synapse that several of these compartments-synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton-labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers and Drosophila shibire(ts1) mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. In shi(ts1) nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show "hot spots" of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.     

Changes in endocrine profile and reproductive organs during puberty in the male marmoset monkey (Callithrix jacchus)

Data on pubertal maturation in male marmoset, a model for human reproduction, are scant and conflicting. We collected data on novel parameters to characterize puberty. Twenty-five marmoset monkeys were assigned to five age groups by weeks (wk): 21 (pre-pubertal), 43 (onset of puberty), 52 (fully pubertal), 70 (mature), and 116 (fully adult). Serum and intratesticular testosterone and pituitary bioactive chorionic gonadotropin (bioCG) were measured. Testicular development was assessed by ultrasonography, histology, and flow cytometry. Three consecutive blood samples revealed extreme fluctuations in testosterone concentrations, suggesting an erratic secretion. Age-related changes in serum testosterone and pituitary bioCG concentrations were observed. Intratesticular androgens (ITAs) showed high fluctuations within groups at all ages and were high in some animals by 21 wk. Unexpectedly, no correlation between pituitary bioCG and serum testosterone or ITAs was found, but these parameters significantly correlated with testicular weight and volume. These observations were consistent a dependence on the testis growth on bioCG. Unfortunately, the low serum levels of bioCG were not measurable in this study. At 43 wk, the animals reached puberty. At 52 wk of age, animals attained maximum body and epididymal weights and qualitatively normal spermatogenesis, but testes continued growing, reaching a maximum of all parameters at 70 wk of age, without further major changes at the age of 116 wk. It is concluded that (1) gonadal activation is evident at wk 21, (2) the male marmoset reaches the pubertal threshold around 43 wk of age, attains qualitative parameters at 52 wk, matures further to sexual maturity at 70 wk, and (3) serum testosterone and ITAs are highly variable without any identifiable correlation with pituitary bioCG.     

Vibrational and thermal study on the in vitro and in vivo degradation of a bioabsorbable periodontal membrane: Vicryl® Periodontal Mesh (Polyglactin 910)

Fourier transform Raman (FT-Raman), attenuated total reflection/Fourier transform infrared (ATR/FT-IR) spectra and differential scanning calorimetry (DSC) measurements were performed on a biodegradable periodontal membrane, the Vicryl® periodontal mesh, in order to study its in vitro and in vivo degradation mechanism and kinetics. The hydrolitic in vitro degradation was investigated in two aqueous media: a saline phosphate buffer (SPB, pH=7.4) and a 0.01 M NaOH solution. Moreover, a membrane implanted in vivo for 4 weeks for treatment of contiguous vertical bony defects, was examined. Vibrational and thermal measurements show that the Vicryl® membrane presents a semicrystalline structure. It degrades faster in the NaOH solution than in the SPB and degradation occurs heterogeneously with a progressive increase in the percentage of crystallinity and shortening of the polymeric chains both in vitro and in vivo. The trends of % weight loss and IR I₆₂₇/I₁₄₁₅ intensity ratio (identified as a marker of crystallinity) are discussed in comparison with the DSC results. The IR I₆₂₇/I₁₄₁₅ intensity ratio and X_c% allow to determine the % weight loss undergone by the membrane degraded in vivo. The result obtained shows that the Vicryl® membrane degrades faster in vivo than in vitro with the formation of oligomers which are more easily absorbed by the surrounding tissues than they are soluble in the degradation media examined.