bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus
Authors: Aldridge Phillip , Paul Ralf , Goymer Patrick , Rainey Paul , Jenal Urs
Published: March 2003

Several members of the two‐component signal transduction family have been implicated in the control of  polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer‐to‐stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant‐negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD∼P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer‐to‐stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole‐specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C‐terminal output domain in modulating the polar cell surface of C. crescentus are discussed.
Fat delays emptying but increases forward and backward antral flow as assessed by flow‐sensitive magnetic resonance imaging
Authors: BOULBY  , MOORE  , GOWLAND  , SPILLER
Published: March 2003

Flow has been assessed in the gastric antrum using a velocity‐sensitive version of the high‐speed magnetic resonance imaging technique, echo planar imaging (EPI). Eight healthy volunteers attended fasted on three separate days and consumed 800 mL of either a 5% glucose (0.2 kcal mL−1), 10% glucose (0.4 kcal mL−1) or an isotonic mixed nutrient meal, FresubinR (1 kcal mL−1, 27.2 g fat). Gastric volumes were obtained at 10‐min intervals for 1 h. Flow measurements were performed on a single slice through the antropyloric region 5 and 35 min after meal ingestion. Gastric volumes at 45 min were inversely proportional to the calorie density of the meal with (mean ± SEM) 89 ± 10%* of the Fresubin, 64 ± 5%* of the 10% glucose and 41 ± 5% of the 5% glucose remaining (*P < 0.005 vs 5% glucose). Substantial forward and backward antral flow was observed after all three meals in the initial 5‐min imaging period. At 35 min flow activity was significantly greater after both the high‐calorie meals relative to the 5% meal (total number of flow events: Fresubin = 6.6 ± 1.7,† 10% glucose = 9.9 ± 2.2,‡ 5% glucose = 2.5 ± 0.9,† P < 0.03,‡ P < 0.007 vs 5% glucose, n = 8). Peak forward velocities for the initial phase of emptying tended to be greater for the rapidly emptying 5% meal (5.9 ± 0.8 cm−1) compared with the Fresubin (3.3 ± 0.6 cm−1, P < 0.069, n = 8) and the 10% glucose (2.9 ± 1.0 cm−1, P < 0.068, n = 8) meals. In spite of delayed gastric emptying, high‐calorie meals were associated with substantial to and fro movements which may be important for meal tritruration and fat emulsification.
Monitoring the lower oesophageal sphincter: sphinctometer or sleeve?
Authors: TRUDGILL  , RILEY
Published: March 2003

Two methods have been used to study lower oesophageal sphincter (LOS) function in gastro‐oesophageal reflux disease: the sleeve sensor and the sphinctometer. Our aim was to directly compare the sleeve and sphinctometer in vivo. Ten asymptomatic volunteers were intubated with a perfused assembly incorporating a sleeve sensor, a solid‐state assembly incorporating a sphinctometer and a pH probe. LOS function was recorded pre‐ and post‐prandially. During basal periods sleeve and sphinctometer readings correlated well both within and between subjects (r2 = 0.89 (P < 0.0001)). However, the sphinctometer relaxed less during swallows (median 42 (interquartile range 27–55)% vs 73 (62–81)% (P < 0.001)), transient LOS relaxations (TLOSR) (50 (40–70)% vs 94 (88–100)% (P < 0.001)) and reflux episodes (67 (59–75)% vs 97 (91–100)% (P = 0.02)). Using criteria derived from 10 dry swallows, the expected relaxation of the sphinctometer during TLOSR was defined. The sphinctometer had an overall sensitivity ranging from 43 to 71% for the detection of TLOSR with 11–22 false positive relaxations, depending on the criteria employed. Sensitivity was particularly poor in those with low basal LOS pressure. The sphinctometer has a lower capacity to register LOS relaxations than the sleeve sensor, which limits its value in studying the pathophysiology of reflux disease.
Effect of gastrin on proximal gastric motor function in humans
Authors: Mearadji  , Straathof  , Lamers  , Masclee
Published: March 2003

The present study was performed to investigate the effect of gastrin on proximal gastric motor and sensory function. Ten healthy volunteers participated in three experiments performed in random order during: (A) continuous intravenous infusion of saline (control) or (B) gastrin (15 pmol kg–1 h–1) reaching postprandial serum gastrin levels or (C) gastrin infusion (15 pmol kg–1 h–1) preceded by acute acid inhibition with intravenous omeprazole. Proximal gastric function was evaluated using a barostat with stepwise pressure and volume distensions and volume measurements during set pressure (MDP + 2 mmHg). Gastrin significantly increased the intragastric volume compared to control during MDP + 2 mmHg (276 ± 39 mL vs. 159 ± 9 mL; P < 0.01) and reduced phasic slow volume wave frequency (from 1.4 ± 0.1 to 0.7 ± 0.1 per min; P < 0.01). During isobaric distensions gastrin increased gastric compliance (42 ± 4 mL mmHg–1 vs. 31 ± 3 mL mmHg–1; P < 0.05). These effects of gastrin infusion were completely abolished by pretreatment with omeprazole. Symptom perception decreased during gastrin infusion and was more dependent on pressure and wall tension than on volume. In conclusion: gastrin may have a role in regulating proximal gastric mechanics by inducing fundic relaxation and increasing gastric wall compliance. The effect of gastrin is dependent on acid secretion.
Alterations of intestinal motor responses to various stimuli after Nippostrongylus brasiliensis infection in rats: role of mast cells
Authors: Gay J. , Fioramonti J. , Garcia‐Villar R. , Buéno L.
Published: March 2003

Nippostrongylus brasiliensis infection induces jejunal mastocytosis associated with enteric nerve remodelling in rats. The aim of this study was to evaluate the intestinal motility responses to meals and to neurotransmitters involved in the control of gut motility (acetylcholine (carbachol), substance P and neurokinin A) in both control and N. brasiliensis‐infected rats 30 days post‐infection. All rats were equipped with NiCr electrodes in the jejunum to record myoelectrical activity. The duration of disruption of the jejunal migrating myoelectrical complexes (MMC) induced by the different stimuli was determined. Meal ingestion and substance P administration disrupted the MMC pattern for similar durations in the two groups. Carbachol and neurokinin A induced a significantly longer MMC disruption in post‐infected rats than in controls (125 ± 8.3 vs. 70 ± 6 min for carbachol 100 μg kg−1 and 51 ± 4 vs. 40 ± 2 for neurokinin A 50 μg kg−1). The enhanced motor response in postinfected rats was reduced by previous mast cell stabilization with ketotifen or mast cell degranulation with compound BrX 537 A. In conclusion, the increased intestinal motor reactivity to carbachol and neurokinin A in post‐N. brasiliensis‐infected rats depends upon intestinal mast cell hyperplasia and degranulation.
Contribution of NK3 tachykinin receptors to propulsion in the rabbit isolated distal colon
Authors: Onori L. , Aggio A. , Taddei G. , Ciccocioppo R. , Severi C. , Carnicelli V. , Tonini M.
Published: March 2003

The role of NK3 receptors in rabbit colonic propulsion has been investigated in vitro with the selective agonist, senktide, and two selective antagonists, SR142801 and SB222200. Peristalsis was elicited by distending a rubber balloon with 0.3 and 1.0 mL of water leading to a velocity of 2.2 and 2.8 mm s−1, respectively. At concentrations of 1 nM, senktide inhibited propulsion evoked by both distensions (range 25–40%), whereas at 6 and 60 nmol L−1 facilitated ‘submaximal’ propulsion by 30%. In the presence of Nω‐nitro‐L‐arginine (L‐NNA, 200 μmol L−1), which per se caused a slight prokinetic effect, 1 nmol L−1 senktide markedly accelerated propulsion (range 35–50%). Hexamethonium (200 μmol L−1) had minor effects on propulsion. In its presence, 60 nmol L−1 senktide significantly inhibited propulsion induced by both stimuli (range 20–50%). SR142801 (0.3, 3 nmol L−1) and SB222200 (30, 300 nmol L−1) facilitated ‘submaximal’ propulsion (range 20–40%). Conversely, higher antagonist concentrations (SR142801: 30, 300 nM; SB222200: 1, 10 μmol L−1) inhibited propulsion to both distensions by 20%. A combination of SR142801 (300 nmol L−1) plus hexamethonium (200 μmol L−1) induced an approximately four‐fold greater inhibition of propulsion than that induced by SR142801 alone. In conclusion, in the rabbit‐isolated distal colon, a subset of NK3 receptors located on descending pathways mediates an inhibitory effect on propulsion by activating a NO‐dependent mechanism. Another subset of NK3 receptors, located on ascen ding pathways mediates a facilitative effect involving a synergistic interaction with cholinergic nicotinic receptors.
Phase III of the migrating motor complex: associated with endogenous xenin plasma peaks and induced by exogenous xenin
Authors: Feurle G. E. , Pfeiffer A. , Schmidt T. , Dominguez‐Munoz E. , Malfertheiner P. , Hamscher G .
Published: March 2003

Xenin, a recently discovered peptide produced by specific endocrine cells of the duodenal mucosa, has shown exocrine, endocrine and motility effects in the gastroenteropancreatic system in animal experiments. The aim of the present investigation was to study the role of xenin in the regulation of duodenojejunal motility of humans. Twenty‐nine healthy volunteers from the hospital staff gave informed consent to participate in this investigation. In 20 volunteers, we determined plasma concentrations of immunoreactive xenin at 15 min intervals over a mean time period of 8 h fasting and recorded the interdigestive motor activity of the duodenojejunum. In a double‐blind randomized crossover study on other nine subjects, synthetic xenin in a dose of 4 pmol kg–1 min–1 or placebo was infused for 10 min intravenously in the interdigestive period and postprandially after a liquid meal. Duodenojejunal motility was recorded simultaneously. Predefined interdigestive xenin plasma peaks were found to be significantly associated with the phases III of the migrating motor complex. In the interdigestive period, xenin induced a premature phase III activity in each volunteer; this was followed by a second phase III in five out of nine subjects. In the postprandial state, xenin significantly increased contraction frequency and the percentage of aborally propagated contractions. These findings suggest a role of the peptide hormone xenin in modulating interdigestive and postprandial duodenojejunal motility in humans.
Free cytosolic Ca2+ recordings from myenteric neurones in multilayer intestinal preparations
Authors: Berghe P. Vanden , Missiaen L. , Bellon E. , Vanderwinden J‐M. , Janssens J. , Tack J.
Published: March 2003

The ability to simultaneously monitor different myenteric neurones in a multilayer preparation may enhance our understanding of the enteric nervous system. Longitudinal muscle myenteric plexus prepar‐ ations were mounted in recording chambers with a coverslip base and loaded with Indo‐1‐AM. cytosolic Ca2+ concentration ([Ca2+]i); changes were recorded at room temperature with a confocal microscope. In addition to mechanical (pressure‐ring) and pharmacological (nifedipine) reduction of muscle contractions, purpose‐designed software was developed to reposition regions of interest and avoid artefacts. Confocal scanning permitted optical selection of single cell layers. High K+ depolarization, used to distinguish between excitable and nonexcitable cells, caused a synchronous [Ca2+]i rise in 84.3% of the ganglion cells. Acetylcholine, substance P and serotonin (all at 10−5mol L−1) induced transient [Ca2+]i changes in subpopulations of myenteric neurones (45.1%, 42.9 and 21.9%, respectively). In addition to immediate responses to agonists, delayed [Ca2+]i changes were also recorded, suggesting the presence of both directly activated and synaptically driven neurones. Functionally identified neurones and other cells in close apposition to the ganglia (interstitial cells of Cajal) could also be studied. This study demonstrates the potential of optical Ca2+ recordings to monitor spread of activity in myenteric neurones and to study their interaction with non‐neuronal targets.
Pharmacological modulation of human gastric volumes demonstrated noninvasively using SPECT imaging
Authors: Liau S‐S. , Camilleri M. , Kim D‐Y. , Stephens D. , Burton D. D. , O'Connor M. K.
Published: March 2003

Three‐dimensional single‐photon emission computed tomography (SPECT) imaging allows noninvasive measurement of human postprandial gastric accommodation. The aim of this study was to determine whether99mTCO4‐SPECT demonstrates effects on pre‐ and postprandial gastric volumes of intravenous (i.v.) erythromycin lactobionate and sublingual isosorbide dinitrate, as predicted from previous literature. Twenty volunteers received no medication (controls), while 12 were randomized to either i.v. erythromycin 2 mg kg−1over 20 min, or 10 mg sublingual isosorbide. After a 10‐min preprandial SPECT measurement, a standard 300‐mL, 300‐kcal liquid meal was ingested, followed by a 20‐min postprandial measurement. Gastric images were reconstructed from transaxial images and total volume was measured using the Analyseð software system. Fasting gastric volume was greater with isosorbide [223 ± 14 (SE) mL vs. 174 ± 9 mL, control; P < 0.05], and postprandial volume was lower with erythromycin [393 ± 27 mL vs. 582 ± 17 mL, control; P < 0.05]. The ratio of postprandial over fasting volume and mean difference between pre‐ and postprandial volumes were significantly lower in both drug groups compared to controls. We conclude that99mTCO4‐SPECT imaging is able to semiquantitatively demonstrate pharmacological modulation of fasting gastric volume and postprandial accommodation in humans.
Validation of a stable isotope gastric emptying test for normal, accelerated or delayed gastric emptying
Authors: Viramontes B. E. , Kim D‐Y. , Camilleri M. , Lee J. S. , Stephens D. , Burton D. D. , Thomforde G. M. , Klein P. D. , Zinsmeister A. R.
Published: March 2003

To validate a 13C‐Spirulina platensis breath test for measurement of accelerated or delayed gastric emptying, we measured gastric emptying of egg containing 13C‐S. platensis and 99mTc‐sulphur colloid by breath 13 CO2 every 15 min over 3 h and scintigraphy every 15–30 min over 5 h in 57 healthy volunteers. Thirty‐three received no treatment, 10 received erythromycin, and 14 atropine. A generalized linear regression model predicted half‐emptying time by scintigraphy (t1/2S) from breath 13CO2 (t1/2B) data. Accuracy was assessed by standard deviation (SD) of differences between t1/2S and t1/2B and by receiver operating characteristic (ROC) curves. Regression models using breath samples at baseline, and 45, 90, 105 and 120 min, predicted t1/2B (mean ± SD) at 118 ± 59 min, similar to t1/2S (118 ± 67 min). Correlation between t1/2B and t1/2S was significant (r=0.88; P < 0.0001). Differences between t1/2S and t1/2B were: 18–19.2 min for t1/2 < 70–150 min, and 68.3 min for t1/2 > l50 min. Breath test detected abnormal emptying with a sensitivity of 86% and specificity of 80%. Thus, the 13C‐S. platensis test measures gastric emptying t1/2 for solids, which is accelerated or delayed to mimic a range of conditions from dumping syndrome to severe gastroparesis, with high sensitivity and specificity. Additional breath samples are needed to increase sensitivity in detecting accelerated gastric emptying.
Unbiased morphometrical measurements show loss of pigmented nigral neurones with ageing
Authors: Ma  , Röytt  , Collan  , Rinne
Published: March 2003

This study used the dissector method to evaluate pigmented nigral neuronal loss in the substantia nigra pars compacta with age. Dissector counts can be used to estimate the absolute and accurate total neurone numbers. In addition, the area and diameter of the neuronal cell body was estimated by using a computerized morphometric analysis in a single section of the substantia nigra pars compacta. Brain samples from 26 people with an age range from 17 to 90 years were studied. A significant decrease in the total number of pigmented neurones (r=−0.83, P<0.001) and their density (r=−0.83, P<0.001) with age was found in the substantia nigra pars compacta. The number of pigmented neurones counted from a single section also showed an age‐dependent decline (r=−0.76, P<0.001). According to the regression equations, the total number of pigmented neurones estimated by dissector counts decreased by 9.8% per decade and the neuronal density decreased by 7.4% per decade. The area of the neuronal cell body decreased by 3.2% per decade. This latter change corresponds to an approximate 4.4% decrease per decade in neuronal volume. These findings show that both the number of pigmented neurones and their size in the substantia nigra pars compacta decreases with age. However, the reductions in the total number of pigmented neurones are more dramatic than the reduction in neurone size with ageing.
p16INK4a and p19INK4d mRNA expression in neuroglial tumours: correlation with Ki67 proliferation index
Authors: Bouvier‐Labit  , Civatte  , Bartoli  , Renaud  , Pellissier  , Figarella‐Branger
Published: March 2003

The INK4a‐ARF locus encodes two unrelated proteins that both function in tumour suppression: p16INK4a and p19INK4d . Although p19INK4d expression has not been studied in central nervous system (CNS) tumours, it has been reported that p16INK4a inactivation is involved in the growth of glioblastomas. This observation has not been reported in relation to other CNS tumours. To understand further the role of p16INK4a and p19INK4d in neuroglial tumour growth, expression of both p16INK4a and p19INK4d mRNAs was studied by reverse transcription polymerase chain reaction RT‐PCR in 59 neuroglial tumours, in which Ki67 labelling indices (LI) were also determined. P16INK4a mRNA was found in all pilocytic astrocytomas (7/7), in all grade II and III astrocytomas (7/7 and 4/4, respectively), in 4/12 glioblastomas, 8/8 oligodendrogliomas, 10/11 anaplastic oligodendrogliomas, 4/7 ependymomas and 3/3 anaplastic ependymomas but not in normal brain. In contrast, p19INK4d mRNA was detected in all tumours and control tissues. p16INK4a expression was associated with a low Ki67 LI in glioblastomas but not in other tumours. P16INK4a expression was not related to anaplasia in oligodendrogliomas and ependymomas. In tumours expressing p16INK4a , in situ hybridization showed a widespread expression of p16INK4a mRNA in tumour cells and in foci of microvascular proliferation. These results strongly support the concept that p16INK4a is involved in the regulation of proliferation in glioblastomas. Other cell cycle regulators which are yet unknown may also play a role in the control of oligodendrogliomas or ependymomas outgrowth. Further studies are required to evaluate the role of p19INK4d in neuroglial tumours.
Repair of the damaged brain. The Alfred Meyer Memorial Lecture 1998
Authors: Dunnett
Published: March 2003

Over the last decade, neural transplantation has progressed from being an experimental technique for studying regeneration and plasticity in the brain to clinical trials of reconstructive surgery in human neurodegenerative disease. Whereas clear evidence is only available at present for the viability of this technique in Parkinson’s disease, applications to several other diseases, including Huntington’s disease, multiple sclerosis, spinal cord injury, and chronic pain are currently under active consideration. It is clear that the techniques of transplantation can be functionally viable under certain well‐defined biological circumstances, but significant problems remain in the availability of suitable donor tissues and defining the optimal conditions for reliable survival of the implanted cells. If we are to obtain improved reliability of the present techniques or identify suitable alternatives, we need a better understanding of the conditions for the survival and integration of grafts into the host brain, and the mechanisms by which they influence host function. In this review I consider the nature of the structural reconstruction required to achieve repair in animal models of Parkinson’s and Huntington’s diseases, contrasting the replacement of deficient neurochemicals within the striatum in the former case, and the need for reconstruction of input and output connections of the striatal circuitry in the latter.
Constitutive and cytokine‐regulated expression of presenilin‐1 and presenilin‐2 genes in human neural cell lines
Authors: Satoh  , Kuroda
Published: March 2003

To investigate the role of pleiotropic neuronal and glial cytokines in the regulation of presenilin (PS) gene expression in human neural cells, both presenilin‐1 (PS1) and presenilin‐2 (PS2) mRNA levels were analysed by Northern blotting in SK‐N‐SH neuroblastoma, IMR‐32 neuroblastoma, NTera2 teratocarcinoma‐derived differentiated neurones (NTera2‐N) and U‐373MG astrocytoma cells following exposure to proinflammatory cytokines (TNF‐α, IFN‐γ, or IL‐1β), anti‐inflammatory cytokines (IL‐10 or TGF‐β1), dibutyryl cyclic AMP or phorbol 12‐myristate 13‐acetate (PMA). The constitutive expression of PS1 (3.0 kb) and PS2 (2.3 kb) mRNA was identified in all these cell lines, in which PS1 mRNA levels were unaltered following treatment with any cytokines and factors examined. By contrast, PS2 mRNA expression was upregulated substantially in SK‐N‐SH cells by exposure to TNF‐α and in U‐373MG cells by treatment with IFN‐γ, whereas it was downregulated in both NTera2‐N and U‐373 MG cells following exposure to IL‐1β or PMA. The levels of PS2 mRNA remained unchanged in IMR‐32 cells after these treatments. These results indicate that PS1 and PS2 genes are expressed constitutively in a panel of human neural cell lines where PS2 mRNA expression is affected by a distinct set of cytokines via cell type‐specific mechanisms that do not alter PS1 mRNA levels, suggesting the existence of separated regulatory systems controlling the expression of PS1 and PS2 genes in human neural cells.
Synapse loss associated with abnormal PrP precedes neuronal degeneration in the scrapie‐infected murine hippocampus
Authors: Jeffrey M. , Halliday W. G. , Bell J. , Johnston A. R. , Macleod N. K. , Ingham C. , Sayers A. R. , Brown D. A. , Fraser J. R.
Published: March 2003

Numbers of neurones, synapses and axon terminals were quantified in a murine scrapie model with severe hippocampal pyramidal cell loss, in which definite clinical scrapie is evident from 226 days post‐infection (dpi) and death occurs around 250 dpi. Disease‐specific PrP accumulations were first seen at 70 dpi (28% of the incubation period (IP)) in thalamus and as sparse foci within the stratum pyramidale of CA1. By 98 dpi (39% IP), PrP was seen in the stratum radiatum and was found at later stages throughout all levels of the hippocampus. At the ultrastructural level in the stratum radiatum of CA1, a decrease in the numbers of simple synapses from 84 dpi (34% IP) and in perforated synapses from 98 dpi (42% IP) was found using an unbiased stereological method, the disector analysis. Degeneration of axon terminals was found from 98 dpi (39% IP) onwards. Neuronal loss was detected in CA1 from 180 dpi (72% IP). The results suggest that the fundamental lesion in the hippocampus of ME7‐infected mice is associated with PrP release from CA1 pyramidal neurones, which perturbs synaptic function and leads to degeneration of preterminal axons, and that subsequent pathological changes including neurone loss are sequelae to this initial insult.
Neuronal death in brain infarcts in man
Authors: Love S. , Barber R. , Wilcock G. K.
Published: March 2003

The mechanism of neuronal death in brain ischaemia remains unclear. Morphology, terminal transferase‐mediated dUTP‐digoxigenin nick end‐labelling (TUNEL) and immunohistochemistry for the pro‐apoptotic enzyme caspase‐3 (CASP3), for its substrates poly(ADP‐ribose) polymerase (PARP) and the DNA‐dependent protein kinase catalytic subunit (DNA–PKCS) and for poly(ADP‐ribose) (PAR), an end‐product of PARP activity, were used to investigate neuronal death in brain infarcts from 15 men and 20 women, aged 46–95 years. The infarcts varied in age from 18 h to several months. Neuronal death was characterized morphologically by cell shrinkage, cytoplasmic hypereosinophilia and moderate nuclear pyknosis with later chromatin dispersal and disintegration, but not features of apoptosis. Occasional apoptotic bodies were seen but these appeared to be related to inflammatory cells, endothelial cells and occasional glia, including satellite cells. Neurones within infarcts showed strong nuclear and cytoplasmic labelling for CASP3 during the first 2 days after infarction. Neuronal DNA–PKCS, PARP and poly(ADP‐ribose) immunoreactivity was demonstrable in scattered neurones in and adjacent to infarcts for 18–24 h but thereafter declined to below detectable levels in most cases. TUNEL labelled cells towards the edge of the infarcts, particularly at 2–4 days, but most of the labelling could be prevented by preincubation of the sections in diethyl pyrocarbonate to inactivate endogenous nucleases. Between 3 days and 3 weeks, CASP3 and DNA–PKCS were detected in proliferating capillaries and CASP3, PARP and poly(ADP‐ribose) in infiltrating macrophages. Our findings indicate that neuronal death in human brain infarcts has some of the early biochemical features of programmed cell death, with upregulation of CASP3 and rapid disappearance of DNA–PKCS and PARP. However, the morphological changes are not those of apoptosis, the DNA cleavage occurs relatively late, and some of the TUNEL is probably mediated by the release of endogenous endonucleases during protease or microwave pretreatment of the damaged tissue.
Ubiquitin C‐terminal hydrolase‐L1 (PGP9.5) expression in human neural cell lines following induction of neuronal differentiation and exposure to cytokines, neurotrophic factors or heat stress
Authors: Satoh J.‐I. , Kuroda Y.
Published: March 2003

Dysfunction of the ubiquitin‐dependent proteolytic pathway contributes to progressive accumulation of ubiquitinated protein inclusions in neurodegenerative disorders, such as Parkinson's disease (PD). Ubiquitin C‐terminal hydrolase‐L1 (UCH‐L1), alternatively designated protein gene product 9.5 (PGP9.5), is a neural deubiquitinating enzyme which is identified as a principal constituent of Lewy bodies. To clarify the regulatory mechanism of UCH‐L1 expression in human neural cells, we studied the constitutive, cytokine/neurotrophic factor‐regulated, and heat stress‐induced expression of UCH‐L1 in cultured human neural cell lines by Western blot analysis. The constitutive expression of UCH‐L1 was identified in SK‐N‐SH neuroblastoma cells, IMR‐32 neuroblastoma cells, U‐373MG astrocytoma cells, and NTera2 teratocarcinoma‐derived differentiated neurones (NTera2‐N). The levels of UCH‐L1 expression were unaltered in these cell lines following treatment with TNF‐α, IL‐1β, BDNF, GDNF, dibutyryl cyclic AMP, or phorbol 12‐myristate 13‐acetate, and remained unchanged by exposure to heat stress. In contrast, its levels were elevated substantially in NTera2 teratocarcinoma cells following retinoic acid‐induced neuronal differentiation, accompanied with an increased expression of α‐synuclein and synaptophysin. These results indicate that UCH‐L1 is expressed constitutively in human neual cell lines, where it is upregulated following induction of neuronal differentiation, but unaffected by exposure to heat stress, cytokines, or growth/differentiation factors which are supposed to be invloved in the nigral neuronal death and survival in PD.
Expression of peripherin in the brain of macaques (Macaca mulatta and Macaca fascicularis) occurs in astrocytes rather than neurones and is associated with encephalitis
Authors: Mathew J. S. , Westmoreland S. V. , Alvarez X. , Simon M. A. , Pauley D. R. , MacKey J. J. , Lackner A. A.
Published: March 2003

Peripherin is a member of the type III intermediate filament family, expressed in neurones of the peripheral nervous system of many species and in a discrete subpopulation of neurones of the central nervous system (CNS) during early development in rodents. Previous studies on rats have shown that peripherin immunoreactivity increased significantly in cell bodies of spinal motor neurones following axonal injury. Our study examined the expression of peripherin in the cerebrum of normal macaques (Macaca mulatta and Macaca fascicularis) and those with encephalitis of viral (simian immunodeficiency virus and simian virus 40) or autoimmune (experimental allergic encephalomyelitis) aetiology. Immunohistochemistry, immunoelectronmicroscopy, immunofluorescence and confocal microscopy were performed on tissue sections using antibodies against cell‐specific markers and peripherin. Peripherin‐positive cells were absent in the cerebrum of normal macaques of all ages examined, whereas animals with encephalitis had peripherin‐positive cells associated with inflammatory infiltrates. Further evaluation revealed that these peripherin‐positive cells were not neurones, but were predominantly astrocytes expressing glial fibrillary acidic protein. Our study suggests that peripherin is not neurone‐specific in the CNS of macaques; peripherin is expressed in astrocytes of animals with encephalitis.
Up‐regulation of MAP2e‐expressing oligodendrocytes in the white matter of patients with HIV‐1 encephalitis
Authors: Cosenza Melissa A. , Zhao Meng‐Liang , Shankar Sai L. , Shafit‐Zagardo Bridget , Lee Sunhee C.
Published: March 2003

HIV‐1 encephalitis (HIVE) is characterized by infection of macrophages and microglial cells, diffuse gliosis, and damage to neuronal populations. The nature of the white matter damage in HIVE remains elusive, and little is known about the status of the oligodendrocyte in HIVE. We have recently described a novel isoform of microtubule‐associated protein‐2 (MAP2e), which is expressed transiently in developing oligodendrocytes during myelination, and in remyelinating oligodendrocytes in multiple sclerosis lesions. In this study, we tested the hypothesis that MAP2e expression would be increased in the white matter of HIVE. We analysed brain sections from patients with HIVE and controls (HIV+ and HIV–) by immunocytochemistry and found that MAP2e+ cells are significantly increased in HIVE (range, 5–167 cells per cm2) compared to controls (range, 1–25 cells per cm2). MAP2e+ cells were negative for GFAP, CD68, LN3, RCA‐1, von Willebrand factor and HIV‐1 p24, but positive for MBP or Luxol‐Fast Blue, supporting their oligodendroglial lineage. A topographical association between MAP2e and HIV‐1 p24 expression was noted, but not between MAP2e and β‐APP, a marker of damaged axons. Our results demonstrate that MAP2e can serve as a marker of white matter damage in HIVE and support the notion that oligodendrocyte damage/repair occurs during HIV‐1 infection.
Risk stratification and pathological mechanisms in preterm delivery
Authors: Lockwood Charles J. , Kuczynski Edward
Published: March 2003

The delivery of infants before 37 weeks gestation is a leading cause of perinatal mortality and morbidity in the United States. Traditional methods of predicting women at risk relying on obstetric history or premonitory symptoms (detected clinically or by tocodynamometry) are neither sensitive nor specific. Recent approaches to predicting preterm delivery have included sonographic measurement of cervical length and various biochemical assays. Although more sensitive than traditional methods, none of these alone exhibits sufficient accuracy to warrant widespread use. We contend that the failure of current approaches to predicting preterm delivery reflects an inadequate understanding of the underlying pathogenesis. Clinical and experimental evidence support the concept that most cases of preterm delivery reflect four pathogenic processes, which share a common final biological pathway leading to uterine contractions and cervical changes with or without premature rupture of membranes. These pathogeneses are: (1) activation of the maternal or fetal hypothalamic‐pituitary‐adrenal axis; (2) decidual‐chorioamniotic or systemic inflammation; (3) decidual haemorrhage (i.e. abruption); and (4) pathological distention of the uterus. Our research seeks to combine the most useful biophysical and biochemical markers of such processes with optimal clinical and epidemiological predictors into a composite, easily applied risk tool. This integrated approach has the potential to identify at‐risk asymptomatic patients with high sensitivity, specificity, and positive and negative predictive values, and also to ascertain underlying pathogenic processes that can lead to targeted therapy. To accomplish these goals, we employ logistic regression and artificial neural network models to assess and apply the appropriate weight to markers associated with each of the above pathogenic pathways, in addition to markers of the final common pathway leading to fetal membrane rupture, cervical extracellular matrix degradation, and myometrial activation.

By combining these markers, we expect ultimately to produce a predictive model that is more robust than any existing method, and that identifies the relative contribution of each pathogenic process. Further analysis of this model using a neural network will enable us to identify asymptomatic patients destined to deliver preterm with high sensitivity, specificity, positive and negative predictive values, and to assess the relative contribution of each of the four distinct pathogeneses to this preterm delivery risk.
Survey on maternal mortality in Swaziland using the sisterhood method
Authors: Lech Medard M. , Zwane Amos
Published: March 2003

Summary

Monitoring and evaluating maternal mortality in African countries is impossible without specific and reliable data and indicators. This study of maternal mortality using the ‘Sisterhood Method’ was undertaken in Swaziland. The crude data on ‘sisterhood mortality’ were obtained from the 1993–94 Multi‐Purpose Household Survey carried out by the Central Statistics Office and Ministry of Health of Swaziland. A total fertility rate of 6.36, as given in the 1986 Swaziland census, was used in estimating these indicators. Prior to this study, the maternal mortality rate (MMR) in Swaziland (based only on health facility data) was considered to lie within the range of 107–125 maternal deaths per 100 000 live births. Use of the ‘Sisterhood Method’ allowed a more precise estimate of maternal mortality for the general population of Swaziland. The study revealed the estimated MMR to be 229 and the life‐time risk of maternal death to be 1 in 69. These values were most probably stable throughout the 6‐ to 7‐year period before the survey.
T cell subset involvement in immune responses to Fasciola hepatica infection in cattle
Authors: McCOLE D.F. , DOHERTY M.L. , BAIRD A.W. , DAVIES W.C. , McGILL K. , TORGERSON P.R.
Published: March 2003

The lymphocyte response to F. hepatica during a primary infection in cattle was analysed to define the role of T cell subsets in the immune response. Blood lymphocytes were isolated from eight cattle infected with F. hepatica via trickle infection over a ten‐day period and from two non‐infected controls. CD4+, CD8+ and γδ+ T cells were depleted from whole lymphocyte populations by magnetic bead depletion. Lymphocytes from infected animals demonstrated a transient, but marked elevation in responsiveness to F. hepatica antigen between weeks 3 and 8 post‐infection. Responses were attenuated by depletion of CD4+ and CD8+ T cells during this period. Depletion of γδ+ T cells attenuated antigen responses at one time point only, and at an earlier stage post‐infection than when αβ+ T cells were depleted. Responses to antigen correlated positively with both hepatic fluke burden and with the degree of hepatic damage. This suggests that the cellular immune response was not protective. Antigen responses in γδ+ T cell‐depleted populations were also associated with post‐mortem fluke burden and with hepatic damage. This suggests that γδ+ T cells are involved in down regulating αβ+ lymphocytes which may have a role in a non‐protective or immunopathological immune responses.
Biochemical characterization and localization of Fasciola hepatica 26‐28 kDa diagnostic coproantigen
Authors: ABDEL‐RAHMAN SALMA , MALONE JOHN B. , O'REILLY KATHY L.
Published: March 2003

We have previously reported the usefulness of a 26–28 kDa coproantigen of Fasciola hepatica for diagnosis of infection. In this study, the 26–28 kDa coproantigen was biochemically characterized with the aid of monoclonal antibodies (MoAb) in an effort to better understand the biology of the antigen. Differential staining of chromatographically‐purified 26–28 kDa coproantigen on SDS‐PAGE, under reducing and non‐reducing conditions, indicated that the coproantigen was a monomeric, highly glycosylated glycoprotein. Alkaline treatment of the purified coproantigen resulted in an 8 kDa protein core which still contained the epitope recognized by the MoAb. No protease activity was associated with the 26–28 kDa coproantigen. The coproantigen could be cleaved by trypsin without altering the reactive epitope recognized by the MoAb, but was resistant to pepsin digestion. Further, the coproantigen was stable under several different storage conditions. Indirect immunofluorescence on tissue sections of adult flukes indicated that the coproantigen was present in gut cells and tegument. Taken together these results confirm the stability of the 26–28 kDa coproantigen and its usefulness in diagnostic tests for F. hepatica infections.
IL‐5 dominates cytokine responses during expression of protective immunity to Onchocerca lienalis microfilariae in mice
Authors: HOGARTH PHILIP J. , BIANCO ALBERT E.
Published: March 2003

In a model of protective immunity against Onchocerca microfilariae (mf), it has been demonstrated previously that immunocompetent mice clear a primary infection and are highly resistant to re‐infection. This immunity correlates with CD4+Th2 cells, is dependant on IL‐5 but not IL‐4, and can be transferred adoptively with spleen cells. In the current investigation, high levels of spontaneous proliferation and of IFNγ production were observed in splenocyte cultures from immune mice, compared with cells from naive controls. Antigen‐specific proliferation also occurred in immune cells, being vigorous following stimulation with adult worm antigen, but not with antigens from developing embryos or mf. Levels of IL‐4, IL‐5 and IFNγ induced by the various antigens was similar, indicating that activation of alternate T helper cell sub‐sets was unlikely to explain the lack of cellular responsiveness. After a primary inoculation with mf, spleen cells from infected mice co‐produced IFNγ and IL‐5. In contrast, IFNγ production was downregulated while IL‐5 levels remained high during active elimination of a challenge infection. Significant levels of IL‐4 production occurred only once parasite clearance had begun. These data confirm the importance of IL‐5 in protection against Onchocerca mf in mice and question the role of IFNγ in the expression of immunity. Production of high levels of IL‐5 correlated with blood and tissue eosinophil mobilization during the clearance of a challenge infection.
Immunogenicity of the extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae delivered by a two‐phase vaccine strain of Salmonella typhimurium
Authors: LATTEMANN  , YAN  , MATZEN ARNE , MEYER  , APFEL HEIKO
Published: March 2003

The recombinant extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae (AVSOD2) was cloned in an expression vector under control of the bacteriophage T7 promoter and the resulting plasmid pLAT7 was introduced in tha aroA attenuated Salmonella typhimurium vaccine strain SL3261::pYZ84. This vaccine strain carries a chromosomally integrated two phase expression system containing inducible T7 RNA polymerase. The recombinant AVSOD2 was efficiently expressed, constituting up to 5% of the total bacterial protein. Furthermore, the plasmid vector containing the AVSOD2 cDNA was shown to be stable over a long period of time in the vaccine strain without antibiotic selection in vitro and in vivo. Jirds which were immunised orally with the recombinant vaccine strain expressing the A. viteae EC‐SOD produced a strong humoral immune response.
The recognition of molecules from fourth‐stage larvae of Ostertagia circumcincta by IgA from infected sheep
Authors: STRAIN  , STEAR
Published: March 2003

The recognition of antigens from fourth‐stage Ostertagia circumcincta by plasma IgA was studied in a group of 30, ten‐month‐old Scottish Blackface sheep which had been naturally, then deliberately infected. Western blotting revealed 49 bands that were recognized by antibody from one or more of the 30 sheep. There was a heterogeneous pattern of antigen recognition amongst the sheep. No individual recognized all the 49 bands and only one of the 49 bands was recognized by all sheep. Two antigens with approximate molecular weights 87 000 Da and 129 000 Da were significantly associated with a reduction in mean adult worm lengths. The observed variation in recognition of these two antigens on fourth‐stage larval preparations accounted, in a statistical sense, for nearly 40% of the total variation in worm length. In addition to the variation in antibody mediated recognition of these two parasite molecules, three other components have been implicated in regulating worm length. They are a 37 000 Da band from adult worms, the amount of fourth‐stage larval specific IgA in the abomasal mucosa and the density‐dependent influence of adult worm burden. Together, these components and their interactions accounted for over 90% of the observed variation in worm length. These results indicate that the parasite‐specific IgA response, or something extremely closely associated with it, is the major immunological mechanism controlling worm length.
Vaccination against Leishmania major in a CBA mouse model of infection: role of adjuvants and mechanism of protection
Authors: Rivier  , Bovay  , Shah  , Didisheim  , Mauël
Published: March 2003

Gp63 is a major surface protein of Leishmania promastigotes. Its protective efficacy has been tested in several experimental models using different mouse strains, gp63 forms, adjuvants and routes of immunization, giving rise to conflicting results. This investigation was designed to determine whether these discrepancies could be ascribed to differing experimental procedures, and to compare gp63‐induced protection with that achieved using live promastigotes. Preliminary experiments demonstrated that gp63 was an extremely potent immunogen compared to a standard antigen (ovalbumin). Protection against Leishmania major infection afforded by gp63 inoculation was studied in CBA mice. Injection of gp63 in saline, or of CFA, BCG, and C. parvum without antigen, induced significant protection. When gp63 and adjuvants were combined, results differed depending on the site of vaccination relative to that of the challenge infection. Vaccination with gp63 plus adjuvants in the tail (i.e. close to the site of infection) led to a stronger reduction of lesion size than the basal level of protection elicited by adjuvants alone, except in the case of CFA. Surprisingly however, when the antigen was injected at a distance from the site of infection (immunization in the hind foot pads, infection in the rump), the protective effect of gp63 was decreased by the adjuvants. Finally, vaccination at either site using live parasites (radioattenuated or virulent promastigotes) resulted in most instances in better protection than achieved by any protocol using gp63 and adjuvants. While anti‐gp63 T cells proliferated in vitro in response to L. major‐infected bone marrow‐derived macrophages, they were unable to activate macrophages for parasite killing. This is in contrast with lymphocytes from mice immunized with live parasites, which both proliferated and stimulated significant killing of the microorganisms within 48 h.
Costimulatory signal through CD86 is important in Th2 response in Trichinella spiralis infected mice
Authors: Wang  , Takamoto  , Sugane
Published: March 2003

In order to study the role of the costimulatory signals in Th2 cytokine production, monoclonal antibodies (mAbs) against cell adhesion molecules (CAMs) were added to cultured cells obtained from the mesenteric lymph nodes (MLNs) of mice infected with Trichinella spiralis, followed by a determination of interleukin (IL)‐5 and IL‐4 in the culture supernatant. IL‐5 production by MLN cells stimulated with somatic antigen was significantly reduced by addition of anti‐CD86 but not by anti‐CD80 mAb. Combination of anti‐CD80 and anti‐CD86 mAbs reduced IL‐5 production most effectively. IL‐4 production induced by anti‐CD3 mAb was suppressed only by the addition of anti‐CD86 mAb. Blockade of the ICAM‐1/LFA‐1 and VCAM‐1/VLA–4 interactions was less effective on the production of IL‐5 and IL‐4 than the addition of anti‐CD86 mAb alone. In contrast to the in vitro cytokine production, intraperitoneal injection of anti‐CD80, anti‐CD86 mAb, or both, similarly suppressed the peak of the eosinophilia on day 21. Elevation of somatic antigen‐specific IgE and IgG1 levels as well as total IgE was not inhibited by the administration of anti‐CD80, anti‐CD86 mAb or both. In‐vitro and in‐vivo effects of CTLA‐4 immunoglobulin were similar to those of combined treatment with anti‐CD80 and anti‐CD86 mAbs. These results suggest that the interaction between antigen‐presenting cells and CD4 T cells through CD86 are most important in Th2 response during T. spiralis infection.