ACE inhibition in goats under fixed-time artificial insemination protocol increases the pregnancy rate and twin births.

To assess the effect of the angiotensin-converting enzyme (ACE) inhibition on the efficiency of the fixed-time artificial insemination (TAI), 69 goats were divided randomly into two groups: enalapril (n = 35) and control (n = 34). In the experiment, all animals underwent the protocol of fixed-time artificial insemination for 12 days. Enalapril group received enalapril maleate dissolved in saline (Enalapril, Lab Teuto Ltda) subcutaneously at the following doses: 0.2 mg/kg/day in D0-D2; 0.3 mg/kg/day in D3-D6 and 0.4 mg/kg/day in D7-D11. The control group received the corresponding volume of 0.9% saline solution. We performed a single insemination 36 hr after sponge removal using frozen semen from two adult male goats with recognized fertility. The ultrasound pregnancy diagnosis was 30 days after the artificial insemination (AI). There was significant increase in pregnancy rates and twinning as well as a decrease in foetal loss in animals receiving enalapril (p < .01). The use of ACE inhibitors during the TAI protocol was shown to be a promising alternative to increase the efficiency of such reproductive biotechnology.

A randomized controlled feasibility trial exploring partnered ballroom dancing for people with Parkinson's disease.

OBJECTIVE: To determine the feasibility of a Dance Centre delivering a programme of mixed dances to people with Parkinson's and identify suitable outcomes for a future definitive trial. DESIGN: A two-group randomized controlled feasibility trial. METHODS: People with Parkinson's were randomized to a control or experimental group (ratio 15:35), alongside usual care. In addition, participants in the experimental group danced with a partner for one hour, twice-a-week for 10 weeks; professional dance teachers led the classes and field-notes were kept. Control-group participants were given dance class vouchers at the end of the study. Blinded assessments of balance, mobility and function were completed in the home. Qualitative interviews were conducted with a subsample to explore the acceptability of dance. RESULTS: A total of 51 people with Parkinson's (25 male) with Hoehn and Yahr scores of 1-3 and mean age of 71 years (range 49-85 years), were recruited to the study. Dance partners were of similar age (mean 68, range 56-91 years). Feasibility findings focused on recruitment (target achieved); retention (five people dropped out of dancing); outcome measures (three measures were considered feasible, changes were recommended). Proposed sample size for a Phase III trial, based on the 6-minute walk test at six months was 220. Participants described dance as extremely enjoyable and the instructors were skilled in instilling confidence and motivation. The main organizational challenges for a future trial were transport and identifying suitable dance partners. CONCLUSION: We have demonstrated the feasibility of conducting the study through a Dance Centre and recommend a Phase III trial.

Candida albicans biofilms formed into catheters and probes and their resistance to amphotericin B.

In Algeria, many bacterial biofilms have been studied but those of fungal origin, particularly those due to the yeast Candida albicans remained unidentified. The present study was performed at the Chabane Hamdoune hospital in Maghnia (Algeria), where 51 strains of C. albicans representing 16.94% of all taken samples were isolated. They were collected from catheters and probes used in different hospital services with variable rates; the most concerned service was ICU (40.74%) followed by gynecology department (17.39%), while general surgery came third (15.79%). Testing the antifungal property of amphotericin B (AmB) we showed clearly that the sessile cells of C. albicans were much more resistant than their planktonic counterparts (suspended cells), especially when the resistance increased during the different phases of biofilm formation until it reached its threshold at the ripening stage (at 48h). Furthermore, scanning electron microscopy of the isolated strains in the laboratory revealed the formation of biofilms on catheters by C. albicans. Surprisingly, observations revealed the presence of a new structure in these biofilms: a chlamydospore?

Impairment of the intestinal barrier is evident in untreated but absent in suppressively treated HIV-infected patients.

BACKGROUND AND AIMS: Impairment of the gastrointestinal mucosal barrier contributes to progression of HIV infection. The purpose of this study was to investigate the effect of highly active antiretroviral therapy (HAART) on the HIV-induced intestinal barrier defect and to identify underlying mechanisms. METHODS: Epithelial barrier function was characterised by impedance spectroscopy and [(3)H]mannitol fluxes in duodenal biopsies from 11 untreated and 8 suppressively treated HIV-infected patients, and 9 HIV-seronegative controls. The villus/crypt ratio was determined microscopically. Epithelial apoptoses were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and caspase-3 staining. Tight junction protein expression was quantified by densitometric analysis of immunoblots. Mucosal cytokine production was determined by cytometric bead array. RESULTS: Only in untreated but not in treated HIV-infected patients, epithelial resistance was reduced (13 (1) vs 23 (2) ohm cm(2), p<0.01) and mannitol permeability was increased compared with HIV-negative controls (19 (3) vs 9 (1) nm/s, p<0.05). As structural correlates, epithelial apoptoses and expression of the pore-forming claudin-2 were increased while expression of the sealing claudin-1 was reduced in untreated compared with treated patients and HIV-negative controls. Furthermore, villous atrophy was evident and mucosal production of interleukin 2 (IL2), IL4 and tumour necrosis factor alpha (TNFalpha) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFalpha and IL13 reduced the transepithelial resistance of rat jejunal mucosa. CONCLUSIONS: Suppressive HAART abrogates HIV-induced intestinal barrier defect and villous atrophy. The HIV-induced barrier defect is due to altered tight junction protein composition and elevated epithelial apoptoses. Mucosal cytokines are mediators of the HIV-induced mucosal barrier defect and villous atrophy.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines.

BACKGROUND: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. METHODS: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2x10(5) cagPAI(-) H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. RESULTS: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. CONCLUSION: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.

Acute lead poisoning in nursing home and psychiatric patients from the ingestion of lead-based ceramic glazes.

To our knowledge, acute inorganic lead poisoning from single ingestions of lead compounds has been only rarely reported. During a 14-month period, we were contacted regarding eight instances of acute ingestions of liquid lead-based ceramic glazes by mentally impaired residents of nursing homes or psychiatric facilities participating in ceramic arts programs. While some ingestions did not cause toxic effects, some patients developed acute lead poisoning characterized by abdominal pain, anemia, and basophilic stippling of red blood cells. In the blood of several patients, lead concentrations were far above normal (4 to 9.5 mumol/L). Urinary lead excretions were tremendously elevated during chelation therapy, with one patient excreting 535.9 mumol/L of lead during a 6-day period, the largest lead excretion ever reported in a patient suffering from acute lead poisoning, to our knowledge. All patients recovered following supportive care and appropriate use of chelating agents. Lead-based glazes are commonly found in nursing homes and psychiatric facilities. We suspect that acute or chronic lead poisoning from the ingestion(s) of lead-based ceramic glazes may be an unrecognized but not uncommon problem among such residents. We urge physicians to take ingestions of lead-based glazes seriously and to consider the diagnosis of lead poisoning in nursing home and psychiatric patients who have participated in ceramic crafts programs.

[Fungal infectivities of implanted catheters due to Candida sp. Biofilms formation and resistance]. / Infectivités fongiques des cathéters implantés dues à Candida sp. Formation des biofilms et résistance.

BACKGROUND: Candidemia are the most common fungal infections in hospitals. However, the catheters are subject to be altered by Candida biofilms which increase the risk of invasive nosocomial infections due to the high resistance to antifungal agents. Therefore, the minimum inhibitory concentrations of planktonic (MIC) and sessile cells (CIMS) were evaluated. METHODS: To review the in vivo biofilms structures of Candida sp. formed on the inner and/or external surfaces of collected catheters, we used scanning electron microscopy (SEM). The level of biofilm resistance was assessed against two conventional antifungal agents: amphotericin B (AmB), which belongs to the class of polyenes, and fluconazole (FLZ) which is an azole. RESULTS: The SEM observation of biofilms of Candida sp. reveals complex structures. Compared to MICs, the calculation of CIMS showed an increase of 32 times with AmB and of 128 times with FLZ. CONCLUSION: Catheters offer an ideal surface to Candida sp. to form biofilms. This complex structure induces the increase of the resistance of sessile cells against two antifungal agents, AmB and FLZ.

Beta amyloid is neurotoxic in hippocampal slice cultures.

We examined the neurotoxicity of the 40 amino acid fragment of beta amyloid peptide (A beta 1-40) in cultured hippocampal slices. When injected into area CA3, A beta 1-40 produced widespread neuronal damage. Injection of the reverse sequence peptide, A beta 40-1, or vehicle alone produced little damage. The distribution A beta 1-40 was highly correlated with the area of neuronal damage. Thioflavine S and electron microscopic analysis confirmed that injected A beta 1-40 formed 7-9 nm AD type amyloid fibrils in the cultures. A beta 1-40 also altered the number of GFAP immunoreactive astrocytes and ED-1 immunoreactive microglia/macrophages within and around the A beta 1-40 deposit. The observed neurotoxicity of A beta 1-40 in hippocampal slice cultures provides evidence that this peptide may be responsible for the neurodegeneration observed in AD.

Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus.

Identified interneurons in the CA1 region of guinea pig hippocampus were examined using light and electron microscopic (EM) techniques. The HRP was intracellularly injected into cells, recorded in the in vitro slice preparation, which met physiological criteria for interneurons. These neurons were characterized at the light and electron microscopic level, and used as standards for identifying interneurons which had not been HRP-labeled. Pyramidal basket cell somata were found in stratum oriens and stratum pyramidale; they were easily identifiable by their convoluted nucleus, dense endoplasmic reticulum, and numerous organelles. Aspinous dendrites reached into stratum oriens, where they received profuse synaptic input (primarily asymmetric synapses). Many of the asymmetric synapses degenerated following commissural lesions, suggesting that much of the input to interneurons was from extrinsic afferents. Dendrites were characterized by their spindled appearance, especially at distal sites. They showed postsynaptic degenerative changes following commissural lesions. Interneuron axons were extremely fine, with regular enlargements or "beads" which made apparent synaptic contacts primarily on pyramidal cell somata. The axon of a single, HRP-injected interneuron made many apparent contacts on large numbers of pyramidal cells; axons arborized over distances of several hundred micra within stratum pyramidale. This study provides direct evidence that neurons, with an identified inhibitory interneuron function in hippocampus, can mediate feed-forward as well as feed-back (recurrent) inhibition. Interneuron output showed extreme divergence, with influence over large distances. The high density of intracellular organelles in these cells suggested high metabolic activity and demand, perhaps making these interneurons exceptionally vulnerable to trauma-induced damage.

Axon arbors and synaptic connections of hippocampal mossy cells in the rat in vivo.

The axon collateralization patterns and synaptic connections of intracellularly labeled and electrophysiologically identified mossy cells were studied in rat hippocampus. Light microscopic analysis of 11 biocytin-filled cells showed that mossy cell axon arbors extended through an average of 57% of the total septotemporal length of the hippocampus (summated two-dimensional length, not adjusted for tissue shrinkage). Axon collaterals were densest in distant lamellae rather than in lamellae near the soma. Most of the axon was concentrated in the inner one-third of the molecular layer, with the hilus containing an average of only 26% of total axon length and the granule cell layer containing an average of only 7%. Ultrastructural analysis was carried out on three additional intracellularly stained mossy cells, in which axon collaterals and synaptic targets were examined in serial sections of chosen axon segments. In the central and subgranular regions of the hilus, mossy cell axons established a low density of synaptic contacts onto dendritic shafts, neuronal somata, and occasional dendritic spines. Most hilar synapses were made relatively close to the mossy cell somata. At greater distances from the labeled mossy cell (1-2 mm along the septotemporal axis), the axon collaterals ramified predominantly within the inner molecular layer and made a high density of asymmetric synaptic contacts almost exclusively onto dendritic spines. Quantitative measurements indicated that more than 90% of mossy cell synaptic contacts in the ipsilateral hippocampus are onto spines of proximal dendrites of presumed granule cells. These results are consistent with a primary mossy cell role in an excitatory associational network with granule cells of the dentate gyrus.

Synaptic connections of dentate granule cells and hilar neurons: results of paired intracellular recordings and intracellular horseradish peroxidase injections.

Simultaneous intracellular recordings were made in the dentate gyrus of rat hippocampal slices, from pairs of the following cell types: granule cells, interneurons located in the granule cell layer, hilar interneurons, and spiny hilar "mossy cells". Granule cells were found to have strong excitatory effects on mossy cells and interneurons. Interneurons inhibited granule cells as well as other interneurons. No synaptic connections from mossy cells onto other cell types were found, within the confines of the slice, using intracellular recording methods. However, at the ultrastructural level, axon terminals of horseradish peroxidase-filled mossy cells were found making synaptic contacts in the hilus on dendrites of interneurons. These studies provide the first step towards determining the functional interactions of the various cell types in the fascia dentata.

Pool cue chalk: a source of environmental lead.

Lead compounds are used as coloring agents for numerous products. Two cases of children with elevated blood lead concentrations encountered by the authors suggested that pool cue chalk may serve as a source of environmental lead. The objective of this study was to determine lead content of various brands and colors of pool cue chalk. Atomic absorption analyses were conducted of 23 different types of pool cue chalk for lead content. Three of 23 types of pool cue chalk contained more than 7000 ppm (mg/kg) lead: one manufacturer's green and tangerine chalk and another manufacturer's green chalk. It was concluded that some brands of pool cue chalk contain relatively large amounts of lead and could contribute to childhood lead poisoning.

Ultrastructure of acetylcholine receptor aggregates parallels mechanisms of aggregation.

BACKGROUND: Acetylcholine receptors become aggregated at the developing neuromuscular synapse shortly after contact by a motorneuron in one of the earliest manifestations of synaptic development. While a major physiological signal for receptor aggregation (agrin) is known, the mechanism(s) by which muscle cells respond to this and other stimuli have yet to be worked out in detail. The question of mechanism is addressed in the present study via a quantitative examination of ultrastructural receptor arrangement within aggregates. RESULTS: In receptor rich cell membranes resulting from stimulation by agrin or laminin, or in control membrane showing spontaneous receptor aggregation, receptors were found to be closer to neighboring receptors than would be expected at random. This indicates that aggregation proceeds heterogeneously: nanoaggregates, too small for detection in the light microscope, underlie developing microaggregates of receptors in all three cases. In contrast, the structural arrangement of receptors within nanoaggregates was found to depend on the aggregation stimulus. In laminin induced nanoaggregates receptors were found to be arranged in an unstructured manner, in contrast to the hexagonal array of about 10 nm spacing found for agrin induced nanoaggregates. Spontaneous aggregates displayed an intermediate amount of order, and this was found to be due to two distinct population of nanoaggregates. CONCLUSIONS: The observations support earlier studies indicating that mechanisms by which agrin and laminin-1 induced receptor aggregates form are distinct and, for the first time, relate mechanisms underlying spontaneous aggregate formation to aggregate structure.

Transgenic animal models for Alzheimer's disease.

The neuropathology of Alzheimer's disease is characterized by the deposition of abnormal protein aggregates. The main constituent of the deposition is beta-amyloid protein. A seminal role of this protein is supported by the discovery of point mutations in the gene of its precursor protein in certain forms of familial Alzheimer's disease. In vitro (cultured neuronal cells), overexpression of the precursor protein or a part of the precursor leads to degeneration of neurons, suggesting neurotoxicity of its derivatives. At this time, all of the reported transgenic mice bearing DNA construct for the precursor or a part of the precursor, however, have not developed convincing pathological changes similar to what is observed in patients with Alzheimer's disease. This interesting discrepancy between in vitro and in vivo suggests suppressors in vivo which ameliorate beta-amyloid precursor protein derivative-mediated neurotoxicity.

Electron microscopy of intracellularly labeled neurons in the hippocampal slice preparation.

We have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron-dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)-labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)-/Neurobiotin (NB)-labeled neurons were recognized by avidin-HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron-dense reaction product via immunocytochemical staining with a rabbit anti-LY antibody, followed by goat anti-rabbit IgG-HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level.

Development of rabbit hippocampus: anatomy.

The postnatal development of the CA1 region of rabbit hippocampus was studied using a variety of light and electron microscopic (EM) techniques. Nissl and Golgi stains showed high cellular density, small cell soma area, and sparse dendritic branching in neurons of immature animals (less than 1 week old); dendritic spines were also relatively infrequent during this period. Cell branching and spine frequency reached near-adult levels by 3 weeks, with the major area of hippocampal expansion seen in the apical dendritic layer. EM examination of synapse patterns was made using osmicated and E-PTA-treated tissue. Both techniques showed that the vast majority of synapses in immature animals (less than 2 weeks old) occurred in the dendritic region and were of the asymmetric type. Axosomatic synapses became less rare by 2 weeks; they were usually of the symmetric synapse type. The pattern of synaptic contacts in immature hippocampus resembled the mature pattern by 3-4 weeks. These data suggest a relatively late development of inhibitory postsynaptic potentials in CA1 pyramidal cells.

Developmental and regional differences in the localization of Na,K-ATPase activity in the rabbit hippocampus.

Regional differences in Na,K-ATPase activity, and development of Na,K-ATPase activity were examined in rabbit hippocampus using a histochemical marker of enzyme activity. Stratum lucidum of CA3/CA2, corresponding to the mossy fiber terminal field, showed high Na,K-ATPase activity compared to stratum radiatum of CA1. A significant increase in Na,K-ATPase activity was found between 8 and 15 days postnatal. Tissues with limited Na,K-ATPase activity (immature hippocampus, the mature CA1 region) appear particularly prone to seizure-like abnormalities, perhaps reflecting an inability to regulate extracellular potassium.

Intracellular dyes mask immunoreactivity of hippocampal interneurons.

The results of several studies have suggested that local circuit neurons, or interneurons, of area CA1 of hippocampus use gamma-aminobutyric acid (GABA) as their neurotransmitter. However, when these cells were labelled by intracellular dye injection, and examined immunocytochemically with antisera raised against GABA, none of the interneurons were immunoreactive. Numerous non-injected interneurons in the same tissue section were clearly immunoreactive. These results suggest that intracellular dyes interfere with immunocytochemical staining of hippocampal interneurons.

Primate neostriatal neurons containing tyrosine hydroxylase: immunohistochemical evidence.

We have detected, in monkey caudate nucleus and putamen, neuronal cell bodies containing tyrosine hydroxylase-like immunoreactivity, as revealed by peroxidase-antiperoxidase immunohistochemistry. Many of these cells are distributed in an outer rim of 1-2 mm throughout the anterior-posterior extent of the neostriatum near its borders with the corona radiata; others are embedded in the adjacent white matter, especially near the ventral putamen and nucleus accumbens. Light and electron microscopy indicate that they are small (8-12 micron), bipolar cells with large nuclei. Such neostriatal neurons, containing tyrosine hydroxylase-like immunoreactivity, number in the tens of thousands.

Changes in gamma-aminobutyric acid and somatostatin in epileptic cortex associated with low-grade gliomas.

The role of specific neuronal populations in epileptic foci was studied by comparing epileptic and non-epileptic cortex removed from patients with low-grade gliomas. Epileptic and nearby (within 1 to 2 cm) non-epileptic temporal lobe neocortex was identified using electrocorticography. Cortical specimens taken from four patients identified as epileptic and nonepileptic were all void of tumor infiltration. Somatostatin- and gamma-aminobutyric acid (GABAergic)-immunoreactive neurons were identified and counted. Although there was no significant difference in the overall cell count, the authors found a significant decrease in both somatostatin- and GABAergic-immunoreactive neurons (74% and 51%, respectively) in the epileptic cortex compared to that in nonepileptic cortex from the same patient. It is suggested that these findings demonstrate changes in neuronal subpopulations that may account for the onset and propagation of epileptiform activity in patients with low-grade gliomas.