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PubMed1995

Transplants of embryonic cortical tissue placed in the previously damaged frontal cortex of adult rats: local cerebral glucose utilization following execution of forelimb movements.

Transplantation of fetal cortical tissue into the motor cortex of adult rats was used as an experimental model to examine the functional integration of homotopic fetal neocortical grafts into the motor pathways of adult host brain. We have employed the [14C]2-deoxy-D-glucose method to analyse the metabolic activity of the transplant and host sensorimotor cortex: (i) in animals solicited to perform specific lever-pressing movements with the limb contralateral to the transplant (experimental group); and (ii) in non-solicited animals or in animals using the limb ipsilateral to the transplant (control group). Grafts in the control group displayed homogeneous uptake of 2-deoxy-D-glucose throughout the rostrocaudal extent of the transplant. The local cerebral glucose utilization levels were low as compared to those of the surrounding cortex but were at least two-times higher than in the corpus callosum. Increase in 2-deoxy-D-glucose uptake by the transplant cells was found only in the experimental group. In this group, 2-deoxy-D-glucose uptake was higher in the caudal (AP: +3.0 to +1.7 mm, relative to Bregma) than in the rostral sectors of the transplants suggesting the existence of a topographic organization within the transplant. In addition, except in the rostral part, glucose utilization was higher in the transplant of the experimental group than in the sensorimotor areas of the non-activated cortex in the control group. Moreover, glucose utilization of the transplant cells was systematically higher in the experimental than in the control group. The transplants appear to display a certain level of metabolic integration with the host sensorimotor cortex since, in the experimental group, there was no significant differences in local cerebral glucose utilization values in the caudal sector of the transplant and in the surrounding sensorimotor cortical areas of the host. The 2-deoxy-D-glucose uptake was even higher in the caudal sector of the transplant than in some of the subfields of the contralateral sensorimotor cortex. The present findings indicate for the first time that motor activation of the contralateral forelimb produces an increase in metabolic activity in distinct transplant sectors, the topographic distribution of which matches the normal topographic organization of the forelimb somatomotor map. This suggests that transplants of embryonic frontal neocortex placed in the frontal cortex of adult hosts become functionally integrated with the host motor system.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMedDate not supplied

ERP evidence of a dichotic left-ear deficit in some dyslexic children.

Children with and without behavioral dichotic left-ear deficits participated in an event-related potential study with quasidichotic presentations of familiar fairy tale segments. Electrical activity was recorded from the scalp while the children listened for semantically and/or syntactically anomalous words from either the right side or the left side while competing segments of the fairy tale were simultaneously presented from the opposite side. Latencies and amplitudes were averaged for each target condition within the group with dichotic left-ear deficits (LED) and the group with normal dichotic listening performance (WNL). Individual global field power waveforms and topographic brain maps were generated for the average response in each of the two listening conditions, target right and target left. Cross-correlations were performed on the grand averaged global field power waveforms to measure the degree of synchrony between target right and target left responses in both groups. Integration functions were performed to compare the accumulated sum of voltages during target (right and left) and control (right and left) conditions. WNL children produced typical ERP responses to the target words in both target right and target left conditions. Responses from LED children were at delayed latencies in the target left condition and were at reduced amplitudes in both target conditions. Topographic brain maps revealed more lateralized scalp distributions and greater activation of frontal regions in LED children in the target left condition. Cross-correlational and integration function results demonstrated interaural asymmetries in responses from the LED children. Overall results suggest that slowed neural conduction times, poor interhemispheric transfer of neural activity, and a failure to suppress competing information arriving at the right ear may be involved in poor left-sided processing in children with behavioral left-ear dichotic deficits.

Analysis of Variance↗

PubMed1985

Biochemical and neuropsychological effects of elevated plasma phenylalanine in patients with treated phenylketonuria. A model for the study of phenylalanine and brain function in man.

Phenylketonuria provides a human model for the study of the effect of phenylalanine on brain function. Although irreversible mental retardation is preventable through newborn diagnosis and dietary phenylalanine restriction, controversy exists regarding the effects of increased concentrations of phenylalanine in older patients. We have studied ten older, treated, phenylketonuric patients using a triple-blind, multiple trials, crossover design. Each patient was tested at the end of each of three 1-wk periods of high or low phenylalanine intakes. Tests included a repeatable battery of neuropsychological tests, analysis of plasma amino acids, and measurement of urine amino acids, phenyl organic acids, dopamine, and serotonin. In all 10 patients plasma phenylalanine rose (900-4,000 microM). In 9 of 10 patients there was an inverse relationship between plasma phenylalanine and urine dopamine excretion. When blood phenylalanine was elevated, these patients had prolonged performance times on neuropsychological tests of higher but not lower integrative function. Urinary serotonin fell during phenylalanine loading in six patients. The concentration of phenylacids in the urine was not proportional to the plasma phenylalanine at concentrations below 1.5 mM. In one patient, neither performance time nor dopamine excretion varied as blood phenylalanine rose or fell. We interpret these data as follows: blood phenylalanine above 1.3 mM impairs performance on neuropsychological tests of higher integrative function, this effect is reversible, and one mechanism may involve impaired biogenic amine synthesis.

Adolescent↗

PubMed2002

Lipid rafts mediate biosynthetic transport to the T lymphocyte uropod subdomain and are necessary for uropod integrity and function.

Polarized migrating T cells possess 2 poles, the uropod protrusion at the rear and the leading edge at the front, with specific protein composition and function. The influenza virus hemagglutinin (HA) is a prototypical molecule that uses lipid rafts for biosynthetic transport to the apical surface in polarized epithelial Madin-Darby canine kidney (MDCK) cells. In this study, HA was used as a tool to investigate the role of lipid rafts in vectorial protein traffic in polarized T lymphocytes. Results show that newly synthesized HA becomes selectively targeted to the uropod subdomain in polarized T lymphoblasts. HA incorporates into rafts soon after biosynthesis, suggesting that delivery of HA to the uropod occurs through a pathway of transport reminiscent of that used for its specific targeting to the apical surface. HA and the adhesion molecules, intercellular adhesion molecule 3 (ICAM-3), CD44, and CD43, 3 endogenous uropod markers, were detected in surface rafts of T lymphoblasts. Cholesterol, a major component of lipid rafts, was predominantly located in the uropod. Disruption of lipid raft integrity by cholesterol sequestration produced unclustering of ICAM-3 and the loss of uropodia and severely impaired processes that require a polarized phenotype such as intercellular aggregation and cell migration. Collectively, these results indicate that lipid rafts constitute a route for selective targeting of proteins to the uropod and that the rafts are essential for the generation, maintenance, and functionality of T-cell anteroposterior polarity.

Antigens, CD↗

PubMed2002

Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea.

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 microg of fGH protein expression per one liter culture containing 1 x 10(8) cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding.

Journal Article↗

PubMed1991

Temporal integration of electrical stimulation of auditory nuclei in normal-hearing and hearing-impaired cat.

Temporal integration functions were measured, before and after a sound-induced hearing loss, in 5 cats using trains of electrical pulses applied to auditory nuclei in the brainstem. The 8 stimuli ranged from 1 pulse (0.25 ms duration) to 16 pulses (0.25 ms pulses spaced over 240 ms). The stimuli were applied to inferior colliculus or cochlear nucleus via permanently implanted electrodes. One electrode was tested extensively in each animal to obtain 10 sets of behaviorally-measured electrical detection thresholds counterbalanced across stimuli. The animal was then exposed to a 110 dB SPL, 2 kHz tone for 48 h and pre- and post-exposure audiograms were measured. The mean permanent threshold shift for acoustic stimuli was 48.5 dB. Another 10 thresholds for each of the 8 electrical stimuli were then measured. In the normal hearing animals, the mean slope of the temporal integration function for electrical stimulation was -7.6 dB per factor of 10 pulses. Alternatively, the mean time constant was 139 ms. In the hearing impaired animals, the slope was reduced to -1.5 dB per factor of 10 pulses, which corresponded to a mean time constant of 17 ms. In addition, the hearing impaired animals showed a decreased threshold for the electrical stimuli (stimulation hypersensitivity) as well as reduced variability across electrical stimulation thresholds. The results suggest that a major contribution to temporal integration occurs in inferior colliculus or higher. In addition, the results suggest that the reduction in temporal integration that follows hearing impairment is a peripherally-induced, central effect.

Animals↗

PubMed1985

Canine liver isolation-perfusion at normo- and hyperthermic temperatures with perfluorochemical emulsion (Fluosol-43).

A perfluorocarbon emulsion, Fluosol-43, was used as a blood substitute for oxygen transport during isolation-perfusion of the dog liver at 37 and 43 degrees C. Preservation of hepatic functional integrity was assessed through analysis of perfusate constituents and animal survival after perfusion. Flow to the liver during perfusion was greater than 1 ml/min/g with one-third of total flow provided through the hepatic artery and two-thirds through the protal vein. Perfusion duration was 3 h. The pO2 gradient across th liver indicated that oxygen was consumed during perfusion at both temperatures. The expected rise in pCO2 and decrease in pH of the outflow perfusate is consistent with active aerobic metabolism. Perfusate chemistries lactate, pyruvate, glucose, urea, total alpha-amino acids, ketone bodies and SGPT demonstrated that hepatic functional integrity was maintained during perfusion. Significant differences (p less than 0.05) between temperatures occurred in the perfusate levels of lactate, pyruvate, L/P ratios, glucose and total alpha-amino acids. Animal survival after a 3-hour perfusion was 3/4 at 37 degrees C, and 2/5 at 43 degrees C. After perfusion, SGPT levels were significantly higher in dogs subjected to perfusion at 43 degrees C. The success of these experiments demonstrates that perfusion of the liver with Fluosol-43 was not in itself hepatotoxic, and that Fluosol-43 may allow perfusion of the liver at 43 degrees C with only wild toxicity.

Alanine Transaminase↗

PubMed1991

GABAA receptor complex function in frontal cortex membranes from control and neurological patients.

The functional integrity of the GABAA receptor-benzodiazepine (BZ) recognition site-Cl- ionophore complex was assessed by means of [35S]TBPS (t-butylbicyclophosphorothionate) binding to frontal cortex membranes prepared from frozen postmortem brain tissue taken from control (n = 4), Alzheimer (n = 7), Parkinson (n = 3) and Huntington's chorea (n = 2) patients. Specific [35S]TBPS binding was similar in control, Parkinson's disease and Huntington's chorea brains, but was significantly reduced (78% control, P less than 0.01) in frontal cortex membranes from Alzheimer's patients. The linkage between the BZ recognition sites and the GABAA receptor-linked Cl- ionophore was functionally intact in these membranes as BZ site agonists (zolpidem, alpidem, flunitrazepam and clonazepam) enhanced [35S]TBPS binding under the conditions used (well-washed membranes in the presence of 1.0 M NaCl). Zolpidem (BZ1 selective) exhibited a biphasic enhancement in control membranes whereas the other compounds induced a bell-shaped concentration-response curve. The enhancement of [35S]TBPS binding by alpidem, flunitrazepam and clonazepam was greater in frontal cortex membranes from Alzheimer's patients than in controls whereas it tended to be reduced in membranes from the brains of Huntington's chorea patients. These studies demonstrate the functional integrity of the GABAA receptor macromolecular complex and also the usefulness of [35S]TBPS binding in the study of human postmortem tissue.

Adult↗

PubMed1993

Intestinal permeability in patients with coeliac disease and relatives of patients with coeliac disease.

The functional integrity of the small bowel is impaired in coeliac disease. Intestinal permeability, as measured by the sugar absorption test probably reflects this phenomenon. In the sugar absorption test a solution of lactulose and mannitol was given to the fasting patient and the lactulose/mannitol ratio measured in urine collected over a period of five hours. The sugar absorption test was performed in nine patients with coeliac disease with an abnormal jejunum on histological examination, 10 relatives of patients with coeliac disease with aspecific symptoms but no villous atrophy, six patients with aspecific gastrointestinal symptoms but no villous atrophy, and 22 healthy controls to determine whether functional integrity is different in these groups. The lactulose/mannitol ratio (mean (SEM) is significantly higher in both coeliac disease (0.243 (0.034), p < 0.0001)) and relatives of patients with coeliac disease (0.158 (0.040), p < 0.005)) v both healthy controls (0.043 (0.006)) and patients with aspecific gastrointestinal symptoms (0.040 (0.011)). The lactulose/mannitol ratio in relatives of coeliac disease patients was significantly lower than in the coeliac disease patient group (p = 0.04). The lactulose/mannitol ratio was the same in healthy controls and patients with aspecific gastrointestinal symptoms. It is concluded that the sugar absorption test is a sensitive test that distinguishes between patients with coeliac disease and healthy controls. The explanation for the increased permeability in relatives of patients with coeliac disease is uncertain. Increased intestinal permeability may be related to constitutional factors in people susceptible to coeliac disease and may detect latent coeliac disease. The sugar absorption test may therefore be helpful in family studies of coeliac disease.

Celiac Disease↗

PubMed2001

Brain function, nonlinear coupling, and neuronal transients.

The brain can be regarded as an ensemble of connected dynamical systems and as such conforms to some simple principles relating the inputs and outputs of its constituent parts. The ensuing implications, for the way we think about, and measure, neuronal interactions, can be quite profound. These range from 1) implications for which aspects of neuronal activity are important to measure and how to characterize coupling among neuronal populations; 2) implication for understanding the emergence of dynamic receptive fields and functionally specialized brain architectures; and 3) teleological implications pertaining to the genesis of dynamic instability and complexity, which is necessary for adaptive self-organization. This review focuses on the first set of implications by looking at neuronal interactions, coupling, and implicit neuronal codes from a dynamical perspective. By considering the brain in this light, one can show that a sufficient description of neuronal activity must comprise activity at the current time and its recent history. This history constitutes a neuronal transient. Such transients represent an essential metric of neuronal interactions and, implicitly, a code employed in the functional integration of brain systems. The nature of transients, expressed conjointly in different neuronal populations, reflects the underlying coupling among brain systems. A complete description of this coupling, or effective connectivity, can be expressed in terms of generalized convolution kernels (Volterra kernels) that embody high-order or nonlinear interactions. This coupling may be synchronous, and possibly oscillatory, or asynchronous. A critical distinction between synchronous and asynchronous coupling is that the former is essentially linear and the latter is nonlinear. The nonlinear nature of asynchronous coupling enables the rich, context-sensitive interactions that characterize real brain dynamics, suggesting that it plays an important role in functional integration.

Animals↗

PubMed1991

Evidence for impaired activation of the hypothalamic-pituitary-adrenal axis in patients with chronic fatigue syndrome.

Chronic fatigue syndrome is characterized by persistent or relapsing debilitating fatigue for at least 6 months in the absence of a medical diagnosis that would explain the clinical presentation. Because primary glucocorticoid deficiency states and affective disorders putatively associated with a deficiency of the arousal-producing neuropeptide CRH can be associated with similar symptoms, we report here a study of the functional integrity of the various components of the hypothalamic-pituitary-adrenal axis in patients meeting research case criteria for chronic fatigue syndrome. Thirty patients and 72 normal volunteers were studied. Basal activity of the hypothalamic-pituitary-adrenal axis was estimated by determinations of 24-h urinary free cortisol-excretion, evening basal plasma total and free cortisol concentrations, and the cortisol binding globulin-binding capacity. The adrenal cortex was evaluated indirectly by cortisol responses during ovine CRH (oCRH) stimulation testing and directly by cortisol responses to graded submaximal doses of ACTH. Plasma ACTH and cortisol responses to oCRH were employed as a direct measure of the functional integrity of the pituitary corticotroph cell. Central CRH secretion was assessed by measuring its level in cerebrospinal fluid. Compared to normal subjects, patients demonstrated significantly reduced basal evening glucocorticoid levels (89.0 +/- 8.7 vs. 148.4 +/- 20.3 nmol/L; P less than 0.01) and low 24-h urinary free cortisol excretion (122.7 +/- 8.9 vs. 203.1 +/- 10.7 nmol/24 h; P less than 0.0002), but elevated basal evening ACTH concentrations. There was increased adrenocortical sensitivity to ACTH, but a reduced maximal response [F(3.26, 65.16) = 5.50; P = 0.0015). Patients showed attenuated net integrated ACTH responses to oCRH (128.0 +/- 26.4 vs. 225.4 +/- 34.5 pmol/L.min, P less than 0.04). Cerebrospinal fluid CRH levels in patients were no different from control values (8.4 +/- 0.6 vs. 7.7 +/- 0.5 pmol/L; P = NS). Although we cannot definitively account for the etiology of the mild glucocorticoid deficiency seen in chronic fatigue syndrome patients, the enhanced adrenocortical sensitivity to exogenous ACTH and blunted ACTH responses to oCRH are incompatible with a primary adrenal insufficiency. A pituitary source is also unlikely, since basal evening plasma ACTH concentrations were elevated. Hence, the data are most compatible with a mild central adrenal insufficiency secondary to either a deficiency of CRH or some other central stimulus to the pituitary-adrenal axis. Whether a mild glucocorticoid deficiency or a putative deficiency of an arousal-producing neuropeptide such as CRH is related to the clinical symptomatology of the chronic fatigue syndrome remains to be determined.

Adrenal Cortex↗

PubMed1989

A comparative in vitro study of low pH and enzyme treated immunoglobulin preparation for intravenous use.

Three not yet commercially available immunoglobulin preparations, intended for intravenous administration, were tested for their purity, physical integrity and in vitro functional activity. Preparations had been factory treated either at pH 4 and with insolubilized pepsin or at pH 4.25 only. Nephelometry revealed a high degree of isotypic purity (IgG greater than 99%). Gel filtration and SDS-PAGE analysis showed only trace amounts of aggregates and/or contaminants, particularly in one of the pepsin treated preparations. Protein A binding inhibition test and phagocytosis of Candida albicans by normal human polymorphonuclear leukocytes, after opsonization of the yeast with different concentrations of immunoglobulin preparations, showed the functional integrity of both the Fc and F(ab)2 regions in the three IgG preparations. Compared to previously reported methods of preparation which produce structural alterations of the Ig molecule, present data indicate the suitability of preparative methods aimed at preserving the integrity of the Ig molecule. Preparation and storage at pH 4.25 without any other treatment appear to be sufficient to obtain pure, aggregate free and in vitro functionally active Ig preparations.

Enzymes, Immobilized↗

PubMed1993

Glaucomatous optic neuropathy causes sensitivity loss to light offsets in the visual field.

The functional integrity of the 'on' and 'off' neurovisual pathways has been examined in the central visual fields of normal (n = 13), glaucomatous (n = 21) and ocular hypertensive (n = 21) individuals. The detection thresholds to light increments (onsets = bright spots) and light decrements (offsets = dark spots) on a uniform background were determined in each eye and the visual field scores were calculated for each of the subjects. Light offsets detected the presence of glaucomatous visual loss and also revealed significant early visual deficits which were missed by the conventional light onset stimuli. This study indicates that 'off-pathway' evaluation with light offsets can be used for the assessment of the functional integrity of the visual system and may reveal sub-clinical early glaucomatous optic neuropathy.

Adult↗

PubMed2003

Maintaining epithelial integrity: a function for gigantic spectraplakin isoforms in adherens junctions.

The Short stop (Shot/Kakapo) spectraplakin is a giant cytoskeletal protein, which exists in multiple isoforms with characteristics of both spectrin and plakin superfamilies. Previously characterized Shot isoforms are similar to spectrin and dystrophin, with an actin-binding domain followed by spectrin repeats. We describe a new large exon within the shot locus, which encodes a series of plakin repeats similar to the COOH terminus of plakins such as plectin and BPAG1e. We find that the plakin repeats are inserted between the actin-binding domain and spectrin repeats, generating isoforms as large as 8,846 residues, which could span 400 nm. These novel isoforms localized to adherens junctions of embryonic and follicular epithelia. Loss of Shot within the follicle epithelium leads to double layering and accumulation of actin and ZO-1 in between, and a reduction of Armadillo and Discs lost within, mutant cells, indicative of a disruption of adherens junction integrity. Thus, we identify a new role for spectraplakins in mediating cell-cell adhesion.

Adherens Junctions↗

PubMed1995

Dopaminergic microtransplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. I. Evidence for anatomical reconstruction of the nigrostriatal pathway.

Reconstruction of the nigrostriatal pathway by long axon growth derived from dopamine-rich ventral mesencephalic (VM) transplants grafted into the substantia nigra may enhance their functional integration as compared to VM grafts implanted ectopically into the striatum. Here we report on a novel approach by which fetal VM grafts are implanted unilaterally into the substantia nigra (SN) of 6-hydroxydopamine (6-OHDA)-lesioned neonatal pups at postnatal day 3 (P3) using a microtransplantation technique. The results demonstrate that homotopically placed dopaminergic neurons survive and integrate well into the previously 6-OHDA-lesioned neonatal SN region. Moreover, the tyrosine hydroxylase (TH)-positive neurons extended axons rostrally along the white matter tract of the internal capsule closely following the course of the original nigrostriatal pathway. The graft reestablished a TH-positive axon terminal network in the ipsilateral caudate-putamen, with the highest density in the medial and central parts. Retrograde labeling with Fluoro-Gold from the host striatum demonstrated that most of the transplant neurons giving rise to the graft-derived fiber outgrowth were TH-positive, but revealed also a small proportion of projecting neurons which were TH-negative. Amphetamine-induced striatal Fos expression was normalized in the caudate-putamen ipsilateral to the intranigral VM grafts, showing hyperexpression in some areas of the striatum, and the apomorphine-induced Fos expression seen in the 6-OHDA-lesioned animals was completely reversed on the grafted side. These findings indicate that the graft-derived dopaminergic reinnervation of the striatum is functional. The microtransplantation strategy may provide new avenues for the exploration of morphological and functional integration of fetal dopamine neurons in the nigrostriatal system and give new insights into the mechanisms controlling long-distance axon growth in the brain.

Amphetamine↗

PubMed2001

The hypothalamic-pituitary-adrenal axis of patients with severe sepsis: altered response to corticotropin-releasing hormone.

OBJECTIVE: To investigate the functional integrity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with severe sepsis by stimulating with corticotropin-releasing hormone (CRH). DESIGN: Prospective observational study in consecutive intensive care unit patients with severe sepsis. SETTING: Surgical intensive care unit and outpatient department of endocrinology in a university hospital. PATIENTS: The study included 20 patients with the diagnosis of severe sepsis; six critically ill, nonseptic patients after major surgery; ten patients with primary adrenal insufficiency; ten patients with anterior pituitary insufficiency; and ten individuals without clinical signs of HPA axis disturbance. INTERVENTIONS: CRH tests were performed with an intravenous bolus injection of 100 microg of human CRH. MEASUREMENTS AND MAIN RESULTS: We studied the functional integrity of the HPA axis in patients with severe sepsis by performing the CRH test. In addition, during the period of severe sepsis, we repeatedly measured basal plasma concentrations of adrenocorticotropin hormone (ACTH) and cortisol. The mean basal plasma cortisol concentration was decreased significantly in nonsurvivors with severe sepsis (288.8 +/- 29.1 [sem] nmol/L) compared with survivors (468.1+/- 18.6 nmol/L; p <.01). By calculating the ACTH/cortisol indices, we found no evidence for adrenal insufficiency in patients with severe sepsis. The mean ACTH/cortisol indices of nonsurvivors with severe sepsis (0.02 +/- 0.008) and survivors (0.01 +/- 0.002) were significantly lower compared with the index of patients with primary adrenal insufficiency (6.8 +/- 1.0; p <.001). In contrast, in nonsurvivors with severe sepsis, the plasma cortisol response to CRH stimulation was impaired compared with survivors: The mean basal cortisol concentration within the CRH test was 269.4 +/- 39.8 nmol/L in nonsurvivors compared with 470.8 +/- 48.4 nmol/L in survivors and increased to a peak value of 421.6 +/- 72.6 nmol/L in nonsurvivors and 680.7 +/- 43.8 nmol/L in survivors (p <.02). However, the change in plasma cortisol, expressed as mean +/- sem and calculated by subtracting the basal cortisol from the peak cortisol after CRH stimulation, was not significantly different in survivors with severe sepsis (243.5 +/- 36.1, range 111.0-524.0 nmol/L, n = 15) compared with nonsurvivors (161.0 +/- 38.9, range 42.0-245.0 nmol/L, n = 5; p >.05). CONCLUSIONS: We found lower basal plasma cortisol concentrations in nonsurvivors compared with survivors of severe sepsis. In addition, the plasma cortisol response to a single CRH stimulation was impaired in nonsurvivors compared with survivors. Reduced responses to CRH stimulation may reflect a state of endocrinologic organ dysfunction in severe sepsis.

Adolescent↗

PubMed1992

Distribution of mast cells in intestinal muscle of nematode-sensitized rats.

We examined the distribution and functional integrity of mast cells in intestinal longitudinal muscle in rats sensitized by two previous infections with Trichinella spiralis. A segment of jejunum was excluded from the gut before infection, and the remainder of the gut was anastomosed. Few mast cells were seen in muscle of noninfected control rats except in the region of the jejunal anastomosis. In rats sensitized by T. spiralis infection, mast cells were increased in number in the jejunum and the number of mast cells followed an aboral gradient down the entire length of the gut in continuity. In addition, mast cells were present in muscle of the excluded segment of sensitized rats. All mast cells were stained red with safranin. Functional integrity was assessed by the ability of mast cells to induce contraction after degranulation by antigen. In muscle from sensitized rats, contraction was induced in each region after exposure to T. spiralis antigen but not Nippostrongylus brasiliensis antigen. Contraction was inhibited by the mast cell stabilizer doxantrazole and the 5-hydroxytryptamine (5-HT) antagonist cyproheptadine. When antigen-induced contraction was expressed as a percentage of the maximum response of the tissue to exogenous 5-HT, the magnitude of contraction decreased along an aboral gradient down the intestine and correlated well (r2 = 0.878) with mast cell numbers. These results suggest that the increase in connective tissue mast cells in gut muscle after T. spiralis infection involves both local and systemic mechanisms.

Animals↗

PubMed2003

Stability of mRNA/cationic lipid lipoplexes in human and rat cerebrospinal fluid: methods and evidence for nonviral mRNA gene delivery to the central nervous system.

Clinical applications of gene therapy require advances in gene delivery systems. Although numerous clinical trials are already underway, the ultimate success of gene therapies will depend on gene transfer vectors that facilitate the expression of a specific gene at therapeutic levels in the desired cell populations without eliciting cytotoxicity. In clinical applications for which transient expression is desirable, mRNA delivery is of particular interest. We have shown cationic lipid-mediated mRNA delivery to be feasible, efficient, and reproducible in vitro. mRNA delivery to the cerebrospinal fluid (CSF) in vivo would provide a means of vector distribution throughout the central nervous system (CNS). This study examined the functional integrity and protection from degradation of mRNA/cationic complexes (lipoplexes) in human cerebrospinal fluid (hCSF) in vitro and expression of these lipoplexes in vivo. Results obtained from gel electrophoresis indicate that cationic lipids protect mRNA transcripts from RNases in hCSF for at least 4 hr. This is in contrast to the total disappearance of nonlipid-complexed mRNA in less than 5 min. We confirmed the importance of RNase activity by incubating mRNA transcripts encoding luciferase or green fluorescent protein (GFP) in hCSF to which RNase inhibitors had been added. After incubation, these solutions were used to transfect Chinese hamster ovary (CHO) cells in vitro. Next, assays for both GFP and luciferase were used to demonstrate functional integrity and translation of the mRNA transcripts. Finally, we delivered in vitro transcribed mRNA vectors encoding for Hsp70 and luciferase to the lateral ventricle of the rat in a series of preliminary in vivo experiments. Initial immunohistochemistry analysis demonstrates that the distribution, uptake, and expression of reporter sequences using lipid-mediated mRNA vector delivery is extensive, as we earlier reported using similar methods with DNA vectors but that the expression may be less intense. Expression was noted in coronal sections throughout the rat brain, confirming the potential for lipid-mediated mRNA delivery to the CNS. These findings confirm that complexing mRNA with cationic lipid before exposure to CSF confers protection against RNase activity, facilitating distribution, cellular uptake, and expression of mRNA delivered into the CNS.

Animals↗