Respiratory rhythms generated in the lamprey rhombencephalon.

Brainstem networks generating the respiratory rhythm in lampreys are still not fully characterized. In this study, we described the patterns of respiratory activities and we identified the general location of underlying neural networks. In a semi-intact preparation including the brain and gills, rhythmic discharges were recorded bilaterally with surface electrodes placed over the vagal motoneurons. The main respiratory output driving rhythmic gill movements consisted of short bursts (40.9+/-15.6 ms) of discharge occurring at a frequency of 1.0+/-0.3 Hz. This fast pattern was interrupted by long bursts (506.3+/-174.6 ms) recurring with an average period of 37.4+/-24.9 s. After isolating the brainstem by cutting all cranial nerves, the frequency of the short respiratory bursts did not change significantly, but the slow pattern was less frequent. Local injections of a glutamate agonist (AMPA) and antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-amino-5-phosphonopentanoic acid (AP5)) were made over different brainstem regions to influence respiratory output. The results were similar in the semi-intact and isolated-brainstem preparations. Unilateral injection of AP5 or CNQX over a rostral rhombencephalic region, lateral to the rostral pole of the trigeminal motor nucleus, decreased the frequency of the fast respiratory rhythm bilaterally or stopped it altogether. Injection of AMPA at the same site increased the rate of the fast respiratory rhythm and decreased the frequency of the slow pattern. The activity recorded in this area was synchronous with that recorded over the vagal motoneurons. After a complete transverse lesion of the brainstem caudal to the trigeminal motor nucleus, the fast rhythm was confined to the rostral area, while only the slow activity persisted in the vagal motoneurons. Our results support the hypothesis that normal breathing depends on the activity of neurons located in the rostral rhombencephalon in lampreys, whereas the caudal rhombencephalon generates the slow pattern.

Morphological and immunohistochemical characterization of interneurons within the rat trigeminal motor nucleus.

Three series of experiments were carried out to characterize interneurons located within the trigeminal motor nucleus of young rats aged 5-24 days. Cholera toxin injections were made bilaterally into the masseter and, sometimes, digastric muscles to label motoneurons. In the first set of experiments, thick slices were taken from the pontine brainstem and cholera toxin-positive and cholera toxin-negative neurons located inside the trigeminal motor nucleus were filled with biocytin through whole-cell recording patch electrodes. Positively identified motoneurons (cholera toxin+) of various shapes and sizes always had a thick, unbranched axon that entered the motor root following a tight zigzag course. Many cholera toxin-negative neurons were also classified as motoneurons after biocytin filling based on this particularity of their axon. These are probably either fusimotor motoneurons or motoneurons supplying other jaw muscles. The cholera toxin-negative neurons classified as interneurons differed markedly from motoneurons in that they had thin, usually branched axons that supplied the ipsilateral reticular region surrounding the trigeminal motor nucleus (peritrigeminal area), the main trigeminal sensory nucleus, the trigeminal mesencephalic nucleus, the medial reticular formation of both sides, and the contralateral medial peritrigeminal area. Most often, their dendrites were arranged in bipolar arbors that extended beyond the borders of the trigeminal motor nucleus into the peritrigeminal area. Immunohistochemistry against glutamate, GABA and glycine was used to further document the nature and distribution of putative interneurons. Immunoreactive neurons were uniformly distributed throughout the rostro-caudal extent of the trigeminal motor nucleus. Their concentration seemed greater toward the edges of the nucleus and they were scarce in the digastric motoneuron pool. Glutamate- outnumbered GABA- and glycine-immunoreactive neurons. There was no clear segregation between the three populations. In the final experiment, 1,1'-dioctadecyl-3,3,3',3'-tetra-methylindocarbocyanine perchlorate crystals were inserted into one trigeminal motor nucleus in thick slices and allowed to diffuse for several weeks. This procedure marked commissural fibers and interneurons in the contralateral trigeminal motor nucleus. Together these results conclusively support the existence of interneurons in the trigeminal motor nucleus.

Anatomical and physiological study of respiratory motor innervation in lampreys.

The innervation of gill muscles of lampreys was investigated in a semi-intact preparation in which the respiratory rhythm was maintained for more than 2 days. Lesion experiments showed that the muscles of gill 1 are innervated by nerves VII (facial) and IX (glossopharyngeal), and those of gill 2 by nerve IX and the first branchial branch of nerve X (vagal). The other gills are supplied by the other branchial branches of nerve X. Retrograde tracers, injected in peripheral respiratory nerves, showed that branchial muscles are innervated by VII, IX and X motoneurons. Within the X nucleus, the motoneuron pools were branchiotopically organized, but with considerable rostro-caudal overlap. Electrophysiological recordings were used to show that the onset of activation of the branchial muscles was increasingly delayed with the distance from the brainstem, but that motoneuronal activity recorded with surface electrodes began at approximately the same time in all pools. The conduction velocity of VII and caudal X motor axons was found to be the same. Differences in the length of motoneuron axons appear to account for the rostro-caudal delay in gill contraction. The data presented here provide a much needed anatomical and physiological basis for further studies on the neural network controlling respiration in lampreys.

Prosthetic valve fungal endocarditis due to histoplasmosis.

Fungal endocarditis is associated with severe patient morbidity and mortality. Unfortunately, fungal endocarditis is difficult to diagnose because fungal pathogens are uncommonly isolated from routine blood cultures. Histopathological examination of surgically excised cardiac valves, peripheral emboli and systemic ulcers may be useful in identifying pathogens as etiological agents of culture-negative endocarditis. The authors describe a 63-year-old man who had culture-negative endocarditis. Multiple echocardiograms showed progression of the vegetations with valve stenosis despite treatment with multiple antimicrobials. He had multiple peripheral emboli before surgery. Disseminated histoplasmosis was diagnosed by bone marrow culture. Yeast organisms consistent with histoplasma were shown in the vegetations of his excised mitral valve prosthesis. The patient was treated with amphotericin and has been doing well in the two years since his surgery. The diagnosis and management of fungal endocarditis are emphasized.

The effects of drafting on stroking variations during swimming in elite male triathletes.

The aim of this study was to determine the effects of drafting behind another swimmer on the metabolic response and stroke characteristics. Six highly trained male triathletes performed two maximal 400-m swims, one in a drafting (D) and one in a non-drafting condition (ND). Their metabolic response was assessed by measuring the oxygen uptake (VO2) and the blood lactate concentration at the end of each 400 m. Swimming velocity, stroke frequency, stroke length, and stroke index (velocity multiplied by stroke length) were recorded every 50 m. In the D and ND conditions, there was no difference in VO2 [66.7 (1.7) ml x kg(-1) x min(-1) vs 65.6 (1.2) ml x kg(-1) min(-1) respectively], however, the lactate concentrations were lower in D than in ND [9.6 (0.9) mM vs 10.8 (0.9) mM, respectively, P < 0.01]. In D, the performance [1.39 (0.02) m x s(-1) vs 1.34 (0.02) m x s(-1), respectively, P < 0.01] and the stroking parameters (i.e., stroke length and stroke index) increased significantly, while the stroke frequency remain unchanged. In D, a stable pace was maintained, while in ND, velocity decreased significantly throughout the 400 m. In D, the performance gains were related to the 400-m D velocity (r = 0.78, P < 0.05), and to the body fat mass (BFM, r = 0.99, P < 0.01). The stroke index in D was also related to BFM (r = 0.78, P < 0.05). Faster and leaner swimmers achieved greater performance gains and stroke index when drafting. Thus, drafting during swimming increases the performance and contributes to the maintenance of stable stroking parameters such as stroke frequency and stroke length during a 400-m swim.

Regional patterning of reticulospinal and vestibulospinal neurons in the hindbrain of mouse and rat embryos.

The dispositions and axonal trajectories of bulbospinal neurons in the pons and medulla of mouse and rat embryos is described from the earliest times these projections can be labelled retrogradely from the cervical spinal cord. Reticulospinal and vestibulospinal neurons are clustered into identifiable groups, each with a characteristic combination of spatial domain and axon trajectory. The various groups can be labelled retrogradely in a specific developmental sequence. The position of some groups shifts from medial to lateral with development, apparently through cell migration. These observations show that the basic regional organization of the reticulospinal and vestibulospinal projections is similar in mouse and rat and is already established during early stages of axon outgrowth.

Hemophagocytic syndrome complicating cardiac transplantation.

Infection-associated hemophagocytic syndrome is one of the hemophagocytic disorders, and is most often seen in the pediatric population, typically in the setting of immunosuppression. We present the case of a 33-year-old man who had been well for more than 3 years following cardiac transplantation until he developed the infection-associated hemophagocytic syndrome. The patient had a fulminant downhill course, dying in shock 10 weeks after his first presentation. Serologic studies for Epstein-Barr virus suggested a remote infection; other viral and microbiologic studies were negative. The only previous report of infection-associated hemophagocytic syndrome complicating cardiac transplant appears to be that of a pediatric patient. The case presented illustrates the difficulties in antemortem diagnosis of this disorder, and in its treatment.

The early neuronal organization predicts the path followed by some major axonal bundles in the embryonic brainstem.

In the embryonic CNS, preformed pathways precede the growth of axonal fasciculi [Katz M. J. and Lasek R. J. (1980) Cell Motil. 1, 141-157; Katz M. J. et al. (1980) Neuroscience 5, 821-833]. What are the developmental events that lead to the elaboration of these preformed pathways? To answer this question, we investigated the organization of the primitive neural tube and more particularly the arrangement of the early-generated cells using [3H]thymidine autoradiography or bromodeoxyuridine. Our data suggest that the position of early-generated cells might be involved in the setting of such pathways. In the brain stem of E12(0) (12 days and 0 h) and E12(15) rat embryos, the first-generated cells were organized into three longitudinal columns associated with glycoconjugate-rich extracellular spaces in the adjacent primitive marginal layer. Also, axons traced with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were contiguous to the early-generated cellular columns and represented the primordium of the medial longitudinal fasciculus, the lateral longitudinal tract and the mesencephalic trigeminal tract. Our results show a correlation between the organization of early-generated cells, likely neurons, and the pattern of extracellular spaces in the marginal layer where axons grow. It has been reported in the literature that neurons produce elements of the extracellular matrix such as growth-modulating molecules or space-creating molecules. We therefore suggest that the position of early-generated neurons could be involved in the elaboration of a template for the setting of some major longitudinal tracts during embryonic development of the brainstem.

Prospective, randomized comparison of intravenous and oral ciprofloxacin with intravenous ceftazidime in the treatment of nosocomial pneumonia.

OBJECTIVE: To compare the efficacy of intravenous and oral ciprofloxacin and intravenous ceftazidime in the treatment of nosocomial pneumonia. DESIGN: Randomized, nonblinded, multicentre comparative trial. SETTING: Seven Canadian university hospitals. POPULATION: Adult patients with moderate to severe pneumonia developing 72 h or longer after hospitalization. METHODS: After informed consent was obtained, patients were randomized to receive intravenous ciprofloxacin 300 mg every 12 h or ceftazidime 2 g every 8 h. After three days, patients in the ciprofloxacin arm could be switched to oral ciprofloxacin, 750 mg every 12 h. Concomitant clindamycin was allowed for three days in patients with syndromes consistent with Gram-positive or anaerobic infection. Erythromycin could be used if cultures revealed no pathogen. RESULTS: A total of 149 patients were enrolled, of whom 124 were eligible for efficacy analysis. Of 119 pathogens identified in 87 patients, 84 were Gram-negative, and 35 Gram-positive. The mean duration of ciprofloxacin therapy was 12.1 days, of which 9.2 days were given intravenously. Ceftazidime was given for a mean of 9.8 days. There was eradication or reduction of pathogens in 75.7% of ciprofloxacin patients and 70.6% of the ceftazidime group. Clinical resolution or improvement occurred in 87.1% of ciprofloxacin recipients and 87.3% of the ceftazidime group. Eight ciprofloxacin and six ceftazidime patients died. Overall outcomes were considered to be successful in 85.2% of ciprofloxacin patients and 87.1% of ceftazidime recipients. Adverse events were mild. CONCLUSIONS: There were similar efficacy and safety of intravenous and oral ciprofloxacin and intravenous ceftazidime in the treatment of patients with hospital-acquired pneumonia. Physicians were reluctant to use oral therapy in patients.

Rhombomere-specific origin of branchial and visceral motoneurons of the facial nerve in the rat embryo.

The goal of this study was to localize selectively the facial nerve branchial and visceral motoneurons in the rat embryo hindbrain. This was achieved by injecting dextran amines into the peripheral facial nerve on embryos maintained in an artificial cerebrospinal fluid. Sprague-Dawley rat embryos 13, 14, and 15 days old (E13, E14, E15) were obtained by cesarean section. Branchial motoneurons were first labeled at E13. They were close to the midline and migrated from rhombomere (r) 4 toward r5 and r6. By E15, they had migrated caudally and ventrolaterally into the former location of r6. Most of them had reached their "adult" position by E15. Another group of motoneurons, the accessory facial nucleus, was found in r4 at E13 and in corresponding regions at later stages. Visceral motoneurons were labeled from the periphery at all stages. At E13, they were mainly in r5 but also in r2, r3, r4, and r6. At E14, most of them had migrated laterally, and, by E15, they were in the prospective parvocellular reticular formation. They could be divided into two subgroups: a more rostral one with fibers that made loops close to the midline and a more caudal one with fibers that went directly to the exit. The findings presented here show that most branchial and visceral motoneurons of the facial nerve are born in different and specific rhombomeres. Interestingly, developmental genes are expressed specifically in these rhombomeres and could be involved in the genesis of the facial and superior salivatory nuclei.

A phase I study of chemically synthesized verotoxin (Shiga-like toxin) Pk-trisaccharide receptors attached to chromosorb for preventing hemolytic-uremic syndrome.

A double-blind, placebo-controlled study was conducted to document possible side effects associated with oral consumption of synthetic verotoxin (VT, shiga-like toxin) Pk-trisaccharide receptor sequences attached to Chromosorb (Synsorb-Pk) by healthy adult volunteers. Synsorb-Pk reclaimed from volunteer stool samples was also analyzed to determine if its VT-binding activity was affected by exposure to the pH extremes and digestive processes of the human gastrointestinal tract. No participant reported any Synsorb-Pk-related adverse reactions, and no clinically important trends in laboratory data were evident. Synsorb-Pk recovered from stools retained its ability to absorb VT in polymyxin extracts of VT-producing Escherichia coli and also neutralized VT when mixed in vitro with VT-positive stools from children with hemorrhagic colitis or hemolytic-uremic syndrome (HUS). These results suggest a potential use for Synsorb-Pk in preventing HUS in patients infected with VT-producing E. coli.

Update on pathogenesis of infective endocarditis.

Specific interactions between pathogens and host factors contribute to the apparent tissue and microbial selectivity in infective endocarditis. Streptococci and staphylococci can produce exopolysaccharides and peptides that have been implicated in adherence to host factors. The presence of a platelet-fibrin matrix on the surface of endothelium can serve as a nidus for colonization by gram-positive cocci, which in turn can promote further aggregation of platelets. Tissue factor expression by valvular endothelial cells is low but can be turned on by endocytosis of staphylococci-this could favor infected thrombus formation. The presence of a foreign body such as a prosthetic heart valve increases the risk of endocarditis. Platelets can promote adherence of staphylococci to foreign body surfaces. Infection of heart valves is the result of influences that in the end will favor microbial attachment and survival. Normal endothelium is resistant to colonization by microorganisms. Antibodies and phagocytes offer some protection against the development of endocarditis. Platelets produce microbicidal proteins that appear important in containing the infection. New diagnostic criteria for endocarditis take into account the pathogenetic characteristics of the disease.

Ontogenetic study of early brain stem projections to the spinal cord in the rat.

In an attempts to describe the early development of the brain stem-spinal projections, we implanted DiI crystals at the C3 level of the spinal cord of 13- and 14-day fixed embryos. After a diffusion period of 2 to 4 months, neurons of the rhombencephalic reticular formation were retrogradely labeled by the tracer. This group of neurons was situated ventromedially in the tegmentum. Their axons coursed into the ventral marginal layer at bulbar levels and entered the ventral funiculus when reaching the spinal cord. Neurons of the lateral vestibular nucleus were also labeled and gave rise to descending fibers that gradually moved medially and entered the spinal cord in the ventral funiculus. In the mesencephalon, labeled cell bodies of the interstitial nucleus of Cajal (InC) were found lying ventrally in the tegmentum, at the rostral end of the medial longitudinal fasciculus (mlf), in which their axons coursed. Also, in the midbrain, several cells lying dorsal to the InC, with axons descending in the lateral tegmentum, were tentatively identified as part of the mesencephalic reticular formation.

The disposition of early-generated neurons in the rat embryo predicts the pattern of major axonal tracts.

During embryogenesis, the fiber tracts grow in a highly stereotyped pattern. A very small number of predetermined paths, preceding the growth of fasciculi, are present in the young neural tube (10-12, 15). What is the origin of these substrate pathways defined by Katz et al. (16) as "... a set of similar guidance cues which are aligned in a continuous discrete pathway..."? Could the first neurons play a role in the guidance of early nerve fibers? Observations in the brain stem revealed the presence of two longitudinal columns of early-generated neurons. These longitudinal columns were associated with well-differentiated marginal zones, characterized by cell-free spaces and representing the prospective site of the medial longitudinal (mlf) and lateral longitudinal (llt) tracts. Nerve fibers were also traced in the brain stem of young embryos. Axons were seen to travel in the early mlf and llt, in close proximity to the regions of early-generated neuronal columns. The data suggest that the precocious neurons that are organized in a definite pattern could somehow be involved in the guidance of some longitudinal axonal tracts, either by directly promoting the formation of an adequate terrain in the marginal layer, or by inducing other cells to do so.

Importance of blood flow to the local distribution of drugs after percutaneous absorption in the bipediculated dorsal flap of the hairless rat.

In order to study the importance of cutaneous blood flow variations to percutaneous drug absorption and local distribution, a bipediculated dorsal flap was created in the hairless rat. This model allowed us to compare the percutaneous drug absorption on both sides of the flap, one with normal blood flow and the other with a reproducible blood flow decrease (60%) obtained by ligation of one pedicle. 14C-labelled compounds, progesterone (lipophilic), caffeine (amphiphilic) and urea (hydrophilic) were applied in ethanolic solution during 4 h on the two sides of the flap. When the cutaneous blood flow was lowered, the skin radioactivity increased greatly for caffeine (76% in superficial dermis, 67% in deep dermis), less for progesterone (37% in superficial dermis, 30% in deep dermis). For urea, no significant difference was observed between the two sides of the flap. Nevertheless, when the absorption of urea was promoted by application on stripped skin, radioactivity was greatly increased on the ligated side of the flap (240% in superficial dermis, 423% in deep dermis). Our results showed that a hydrophilic compound poorly absorbed (urea) was insensitive to cutaneous blood flow modifications whereas compounds readily absorbed (caffeine, amphiphilic; urea on stripped skin) or slightly absorbed (progesterone, lipophilic) exhibited local concentration phenomena in relation to cutaneous blood flow lowering.

Interaction of Shigella toxin with globotriaosyl ceramide receptor-containing membranes: a fluorescence study.

The interaction of the B-subunit of Shigella toxin with a globotriaosyl ceramide receptor incorporated into phosphatidylcholine vesicles was studied by fluorescence spectroscopy. From the position of the maximum in the emission spectrum and the accessibility to acrylamide quenching, it is concluded that a single tryptophan of a free B-chain is located in a highly polar environment, most likely on the surface of the folded polypeptide chain. Binding of B-subunits to the membrane-associated globotriaosyl ceramide results in a strong enhancement of fluorescence intensity and a small blue-shift of the emission maximum; these effects suggest a conformational change in the protein which provides a new environment to a tryptophan residue. However, the polarity of this new environment is still relatively high--as indicated by the position of the emission maximum at 344 nm--and suggests that the receptor-bound B-chain remains largely on the membrane surface, without penetrating the hydrophobic interior of a lipid bilayer. On the other hand, the A-chains are shown to interact directly with the receptor-free lipid bilayers; this nonspecific interaction may play a role in the mechanism by which A-subunit traverses the membrane of a target cell.

A new model: bipediculated dorsal flap of hairless rat for cutaneous blood flow evaluation.

To study the influence of cutaneous blood flow upon the transdermal penetration of drugs, an original in vivo model, the bipediculated dorsal flap (BDF) of the hairless rat is described. This model is constituted by a dorsal cutaneous muff on both sides of the spinal column. The blood circulation was maintained symmetrically on both skin insertion pedicles. Two radiotracers, 86Rb and 201Tl (respective half-lives: 18 and 3 days), known to measure nutritional blood flows, are compared in this model. In the back skin, over and under the flap, mean blood flow (expressed as percentage of injected dose per gram skin) was similar with Rb (9.5 +/- 3.1) x 10(-2) and Tl (10.2 +/- 2.1) x 10(-2). Equivalent values were obtained in BDF skin (non ligated pedicle): Rb (8.4 +/- 1.3) x 10(-2), Tl (9.5 +/- 2.7) x 10(-2). In BDF skin, cutaneous blood flow was lower by 70-91% in the ligated pedicle (compared to the nonligated) for both radioisotopes. Identical 86Rb and 201Tl distributions suggest the use of 201Tl for further experiments of cutaneous blood flow.

Nucleotide sequence of the Shiga-like toxin genes of Escherichia coli.

We have determined the nucleotide sequence of the sltA and sltB genes that encode the Shiga-like toxin (SLT) produced by Escherichia coli phage H19B. The amino acid composition of the A and B subunits of SLT is very similar to that previously established for Shiga toxin from Shigella dysenteriae 1, and the deduced amino acid sequence of the B subunit of SLT is identical with that reported for the B subunit of Shiga toxin. The genes for the A and B subunits of SLT apparently constitute an operon, with only 12 nucleotides separating the coding regions. There is a 21-base-pair region of dyad symmetry overlapping the proposed promoter of the slt operon that may be involved in regulation of SLT production by iron. The peptide sequence of the A subunit of SLT is homologous to the A subunit of the plant toxin ricin, providing evidence for the hypothesis that certain prokaryotic toxins may be evolutionarily related to eukaryotic enzymes.

Complete amino acid sequence of Shigella toxin B-chain. A novel polypeptide containing 69 amino acids and one disulfide bridge.

The complete amino acid sequence of the B-chain of Shigella toxin has been determined using both liquid- and gas-phase sequenators. It reveals a 69-amino acid peptide with a single disulfide bridge, predicting a subunit molecular weight of 7691. No Asn-X-Ser(Thr) sequence was found, confirming the absence of potential N-glycosylation sites. A computer data bank search using a mutation data matrix did not detect any similarity greater than 30% with known sequences to date, indicating a novel primary structure. However, some distant homology with the 103-residue B-chain of cholera and Escherichia coli enterotoxins was revealed. Hydropathy, fractional exposure, and Chou and Fasman calculations all point to an ordered structure with a hydrophobic core spanning residues 36-52 and a hydrophilic domain between residues 10 and 20, the latter probably representing the most antigenic domain.