Klebsiella pneumoniae resistant to third-generation cephalosporins in five African and two Vietnamese major towns: multiclonal population structure with two major international clonal groups, CG15 and CG258.

The molecular epidemiology of third-generation cephalosporin-resistant (3GC-R) Klebsiella pneumoniae in developing countries is poorly documented. From February 2007 to March 2008, we collected 135 3GC-R K. pneumoniae isolates from seven major towns in Maghreb (Morocco), West Africa (Senegal, Côte d'Ivoire), Central Africa (Cameroon), East Africa (Madagascar) and Southeast Asia (Vietnam). Their genetic diversity, assessed by multilocus sequence typing, was high (60 sequence types), reflecting multiclonality. However, two major clonal groups, CG15 (n = 23, 17% of isolates) and CG258 (n = 18, 13%), were detected in almost all participating centres. The two major clonal groups have previously been described in other parts of the world, indicating their global spread. The high diversity of enterobacterial repetitive intergenic consensus sequence-PCR banding patterns at the local level indicates that most isolates were epidemiologically unrelated. The isolates were characterized by the presence of multiple resistance determinants, most notably the concomitant presence of the aac(6')-Ib-cr, qnr and blaCTX-M-15 genes in 61 isolates (45%) belonging to 31 sequence types. These isolates were detected across a large geographical area including Cameroon (n = 1), Vietnam (n = 4), Madagascar (n = 10), Côte d'Ivoire (n = 12), Morocco (n = 13) and Senegal (n = 21). These results have major implications for patient management and highlight a potential reservoir for resistance determinants.

Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration.

Klebsiella pneumoniae BM2974 isolated from an abdominal abcess was resistant to high concentrations of all available beta-lactams, including recently developed third-generation cephalosporins and carbapenems. Isoelectric focusing of beta-lactamases and amplification, cloning and sequencing of the corresponding genes, together with conjugation and transformation experiments, indicated that, in addition to the chromosomally encoded beta-lactamase, the strain produced three plasmid-mediated beta-lactamases with pIs of 5.4, 8.2 and 9.0, which corresponded to TEM-1, SHV-5 and AmpC-type CMY-4, respectively. Strain BM2974 also lacked a major outer membrane protein of c. 40 kDa which was present in the spontaneous imipenem-susceptible revertant BM2974-1. We suggest that imipenem resistance in strain BM2974 is attributable to production of CMY-4 beta-lactamase combined with permeability alteration.

Developmental changes in nicotinic receptor mRNAs and responses to nicotine in the suprachiasmatic nucleus and other brain regions.

Our previous studies demonstrated that nicotine induces c-fos expression in the suprachiasmatic nucleus (SCN) of the rat during a narrow developmental window occurring in the perinatal period. We have extended these observations by showing that c-fos cannot be induced in the adult SCN by nicotine even during the subjective night, when phase shifts do occur. In contrast to the SCN, significant induction of c-fos and NGFI-A was observed in the medial habenula and paraventricular nucleus at all circadian times. In the fetal rat SCN we show that NGFI-A and junB are also induced by nicotine, but not c-jun. To investigate whether changes in nicotinic acetylcholine receptor (nAChR) expression in the SCN may underlie this change in sensitivity during the perinatal period, we examined nAChR mRNAs across this developmental period. By Northern analyses, alpha2, alpha3 and alpha4 subunit mRNAs are relatively abundant in the fetal SCN but decline substantially in the adult. alpha7 mRNA increases substantially while beta2 mRNA is relatively abundant throughout development. We also examine expression in the whole mouse brain beginning at embryonic day 11. Many mRNA sizes for nAChR subunits in both the rat and mouse are characterized here for the first time by Northern analyses and some show very large changes in expression across development. In particular, a small 1.4 kb alpha2-related mRNA is highly expressed during early development, perhaps indicating an important novel function for this subunit.

The glutamate induced phase shift in the SCN slice: a two pulse study.

The short-term dynamics of resetting the circadian 'clock' was assessed by a double-pulse paradigm in vitro. On day 1, single and double 1 h 'pulses' of 1 mM l-glutamate were applied to the rat suprachiamastic nuclei (SCN). On days 2 and 3, single unit activity (SUA) was recorded and time-of-peak SUA was used as a phase marker of the circadian rhythm. The time-of-peak in untreated slices at 'Zeitgeber' time (ZT; hours after lights-on) 6, was used to evaluate effects of glutamate on phase. In accordance with published data, a single glutamate pulse at ZT 14 resulted in a 3 h delay of peak SUA on days 2 and 3. A 2nd pulse, given 3 h after a 1st pulse, resulted in two distinct peaks on day 2: a 1st at ZT 7 and a 2nd at ZT 12, i. e., a 6 h phase delay and hence twice the delay obtained after a single pulse. On day 3, no peak in SUA was observed which indicates that a new steady state was not reached on day 2. The bimodal distribution of SUA on day 2 corroborates other findings which suggest that the SCN comprises two distinct neuronal populations with circadian firing patterns that are normally coupled but, possibly due to different sensitivities to glutamate, can desynchronize. The additive phase-shifting effect of two consecutive glutamate pulses suggests that, at least for one sub-population of SCN neurons, the phase shift is completed within 3 h.

Circadian system of mice integrates brief light stimuli.

Light is the primary sensory stimulus that synchronizes or entrains the internal circadian rhythms of animals to the solar day. In mammals photic entrainment of the circadian pacemaker residing in the suprachiasmatic nuclei is due to the fact that light at certain times of day can phase shift the pacemaker. In this study we show that the circadian system of mice can integrate extremely brief, repeated photic stimuli to produce large phase shifts. A train of 2-ms light pulses delivered as one pulse every 5 or 60 s, with a total light duration of 120 ms, can cause phase shifts of several hours that endure for weeks. Single 2-ms pulses of light were ineffective. Thus these data reveal a property of the mammalian circadian clock: it can integrate and store latent sensory information in such a way that a series of extremely brief photic stimuli, each too small to cause a phase shift individually, together can cause a large and long-lasting change in behavior.

Nicotine and nicotinic receptors in the circadian system.

Considerable data support a role for cholinergic influences on the circadian system. The extent to which these influences are mediated by nicotinic acetylcholine receptors (nAChRs) has been controversial, as have the specific actions of nicotine and acetylcholine in the suprachiasmatic nucleus (SCN) of the hypothalamus. In this article we review the existing literature and present new data supporting an important role for nAChRs in both the developing and adult SCN. Specifically, we present data showing that nicotine is capable of causing phase shifts in the circadian rhythms of rats. Like light and carbachol, nicotine appears to cause phase delays in the early subjective night and phase advances in the late subjective night. In the isolated SCN slice, however, only phase advances are seen, and, surprisingly, nicotine appears to cause the inhibition rather than the excitation of neurons. Among nAChR subunit mRNAs, alpha 7 appears to be the most abundant subunit in the adult SCN, whereas in the perinatal period, the more typical nAChRs with higher affinity for nicotine predominate in the SCN. This developmental change in subunit expression may explain the dramatic sensitivity of the perinatal SCN to nicotine that we have previously observed. The effects of nicotine on the SCN may contribute to alterations caused by nicotine in other physiological systems. These effects might also contribute to the dependence properties of nicotine through influences on arousal.

Diagnosis of enteric fever caused by Salmonella spp. in Vietnam by a monoclonal antibody-based dot-blot ELISA.

Enteric fever caused by Salmonella spp. is prevalent in Vietnam. None of the currently available diagnostic methods meets the ideal criteria on rapidity, simplicity, sensitivity, specificity, cost-effectiveness and practicality for developing areas. In this study, a recently developed monoclonal antibody-based dot-blot ELISA was used in comparison with the hemoculture method and the classical Widal test for diagnosis of salmonellosis in 171 Vietnamese patients presenting with clinical features of enteric fever. Urine samples of 50 healthy counterparts were used as negative controls. Salmonella spp. were isolated from 77 of 171 patients (45%) while 98 and 111 patients were positive by dot-blot ELISA and Widal test, respectively. The diagnostic sensitivity, specificity, accuracy, positive predictive value and negative predictive value of the ELISA performed on three serial urine samples collected at 2 hour intervals of the 171 patients were 92.2%, 71.3%, 80.7%, 72.4% and 91.8%, respectively when compared with the culture method. The Widal test performed on acute and convalescence serum samples showed 87.0%, 46.8%, 68.4%, 60.4% and 83.3% diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values, respectively when compared with the bacterial culture method. Kappa coefficience revealed very good agreement beyond chance between the MAb-based ELISA and the culture method. The ELISA was not reactive when tested on urine samples of 50 healthy individuals which indicates 100% specificity. The Salmonella antigenuria of the patients as detected by ELISA lasted 10.3+/-3.9 days after initiating antibiotic treatment. The MAb-based dot-blot ELISA is easy to perform. It is rapid, sensitive, specific, inexpensive, and non-invasive and does not require equipment, thus is suitable for developing areas. It can detect acute/recent infection and can be used for evaluation of the efficacy of the treatment.

Dopamine enhancement and depression of glutamate-regulated calcium and electrical activity in hypothalamic neurons.

1. The neurotransmitter dopamine is found throughout the hypothalamus both in cell bodies and in axons originating from intra- and extrahypothalamic sources. To study the mechanisms of action of dopamine on cultured rat hypothalamic neurons, particularly in relation to Ca2+ regulation, we used Ca2+ digital imaging with fura-2 and whole cell patch-clamp recording. We focused on the modulatory actions of dopamine on glutamate. 2. Dopamine administration had little or no independent effect on intracellular Ca2+. However, in the presence of tetrodotoxin to block action potentials and action-potential-dependent transmitter release, dopamine (10 microM for 2-3 min) caused an increase in glutamate-evoked Ca2+ rises in 22% of 64 neurons and depressed glutamate-evoked Ca2+ rises in an equal number of neurons. Shorter exposure to dopamine reduced the number of responding cells. 3. Dopamine application to neurons with an elevated Ca2+ due to synaptic release of glutamate (in the absence of tetrodotoxin) generally caused a decrease in Ca2+ levels (40% of 106 neurons), but sometimes increased cytosolic Ca2+ (10% of 106 neurons). That dopamine influenced cells differently in conditions of spontaneous activity compared with evoked activity may be due to dopamine effects on presynaptic receptors detected under conditions of ongoing synaptic release of glutamate. 4. Dopamine modulation of glutamate responses was detected at early stages of neuronal development (embryonic day 18 after 2 days in vitro) and also after 60 days in vitro. 5. The D1, D2, and D3 dopamine receptor agonists SKF38393, quinpirole, and 7-OH-DPAT (+/- 7 hydroxy-dipropylaminotetralin) caused a reduction in Ca2+ levels raised by endogenous glutamate release or evoked by exogenous glutamate application. 6. To block the actions of dopamine released by hypothalamic neurons, D1 and D2 dopamine receptor antagonists were used. As with dopamine, dopamine antagonists had no effect on intracellular Ca2+ during glutamate receptor blockade. In the absence of glutamate receptor block, the D1 antagonist SCH23390 (1 microM) reduced Ca2+ in responding cells; in contrast, the D2 antagonist eticlopride (1 microM) generated a delayed increase in Ca2+ levels. 7. Dopamine is known to activate second messengers through G proteins independent of changes in membrane potential or input resistance. Whole cell recording was used to demonstrate that, parallel to the modulation of Ca2+, dopamine exerted a dramatic change in glutamate-mediated electrical activity, generally depressing activity and hyperpolarizing the membrane potential (8 of 15 neurons). In a smaller number of neurons (5 of 15), dopamine enhanced glutamate-mediated excitatory activity. 8. Dopamine-evoked changes in membrane potential were in part mediated through modulation of glutamate actions. Dopamine depressed glutamate-evoked currents in a dose-dependent fashion, with Hill slopes in individual neurons ranging from 0.3 to 0.6. Dopamine could also evoke a direct hyperpolarizing action on hypothalamic neurons in the presence of tetrodotoxin or glutamate receptor blockers, at least in part by opening K+ channels. 9. Glutamate plays an important role as a primary excitatory transmitter within the hypothalamus. Our data support the hypothesis that a major mechanism of dopamine's influence on hypothalamic neurons involves the modulation of glutamate's excitatory action, mostly by inhibition. This is consistent with the hypothesis that modulation of glutamate activity may be an important mechanism of dopamine action throughout the nervous system.

Embryonic hypothalamic expression of functional glutamate receptors.

Glutamate can play a number of roles in the developing brain, including modulation of gene expression, cell motility, neurite growth and neuronal survival, all critical for the final organization and function of the mature brain. These functions are dependent on the early expression of glutamate receptors and on glutamate release in developing neurons. This subject has received little attention in the hypothalamus, despite glutamate's critical role as an excitatory transmitter in hypothalamic control of circadian rhythms, endocrine secretion, temperature regulation, and autonomic control. A total of 10,922 rat hypothalamic neurons were studied with digital Ca2+ imaging with the ratiometric dye fura-2 to examine their responses to glutamate receptor agonists and antagonists during embryonic development and maturation in vitro. Functional glutamate receptors were found very early in development (embryonic day 15-E15) with both Ca2+ imaging and with patch clamp recording. This is a time when the hypothalamus is beginning to undergo neurogenesis. Ca2+ responses from N-methyl-D-aspartate receptors developed later than those from non-N-methyl-D-aspartate ionotropic receptors that responded to kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate. The responses of immature E15 cells after one day in vitro were compared with more mature cells after six days in vitro to examine the response to repeated 3 min applications of 100 microM kainate (n = 108). Immature cells showed similar Ca2+ rises (+232nM Ca2+) with each kainate stimulation. In contrast, more mature cells showed an initial Ca2+ rise of 307 nM, with the second rise only to 147 nM above the initial baseline. Immature cells more quickly returned to their pre-kainate baseline than did older cells. The expression of metabotropic glutamate receptors was studied with the selective agonist trans-1-amino-cyclopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five days in vitro. E16 astrocytes showed a greater response than did neurons to conditions that would activate the metabotropic glutamate receptor. A dramatic increase in the percentage of cells that responded to N-methyl-D-aspartate was found after only a few days in culture. Only a small number of E15 cells studied on the day of culture (4% of 694 cells) showed a response to 100 microM N-methyl-D-aspartate. Thirty-eight percent of 120 E18 cells cultured for one day in vitro showed an N-methyl-D-aspartate response.(ABSTRACT TRUNCATED AT 400 WORDS)

Thermal effects on neuronal activity in suprachiasmatic nuclei of hibernators and nonhibernators.

The temperature sensitivity of neuronal firing rates in the suprachiasmatic nuclei (SCN) of the hypothalami of rats and ground squirrels was studied in vitro. SCN from euthermic squirrels were studied during the hibernation season (winter) and during the summer. SCN from hibernating squirrels were also studied. Most properties of SCN cells from hibernators and nonhibernators were similar. Warm- and cold-sensitive neurons were observed in all groups, but cold-sensitive neurons were more common in SCN from hibernating squirrels. No evidence for temperature compensation of firing rate was accumulated; no cell was observed to fire below 16.6 degrees C. If the persistence of circadian rhythmicity is a function of action potential-dependent neurotransmission from the SCN, these results suggest that deep hibernation (5-17 degrees C) should be characterized by an absence of circadian fluctuation in temperature. Two possible adaptations for the shallow torpor seen at somewhat higher temperatures were observed in the SCN: 1) a relatively large population of cold-sensitive neurons and 2) a population of neurons with very high activation energies. Activation energy analysis suggested that most of the temperature-sensitive properties of these cells could be explained in terms of the thermal sensitivity of the sodium channel.

Saturable efflux of the peptides RC-160 and Tyr-MIF-1 by different parts of the blood-brain barrier.

Peptides have been shown to be transported in the direction of both blood to brain and brain to blood. Although blood to brain transport is known to occur at both the choroid plexus and the capillary bed of the brain, comprising the two major components of the blood-brain barrier, the location of efflux systems for peptides remains largely unstudied. We adapted established methodologies to study this question for two peptides known to be transported out of the brain after injection into the cerebrospinal fluid (CSF): Tyr-MIF-1, transported by peptide transport system (PTS)-1 and RC-160, a somatostatin analog transported by PTS-5. Radioactive iodide, known to be transported out of the brain primarily by the capillaries, also was studied. We found that after injection into brain tissue, RC-160 and iodide were rapidly transported out of the brain by saturable mechanisms. By contrast, efflux of Tyr-MIF-1 was slow and nonsaturable after injection into brain tissue, but rapid and saturable after injection into the lateral ventricle of the brain. Autoradiography confirmed that peptide injected into brain tissue did not diffuse far from the site of injection during the study period. The results indicate that the efflux system for RC-160 is located at least partly at the capillaries and suggest that the major location for the efflux system of Tyr-MIF-1 is at the choroid plexus.

Correlation between the levels of antinuclear antibodies, anti-DNA antibodies, and complement in systemic lupus erythematosus.

Antinuclear antibody (ANA) titres, anti-native DNA levels, serum haemolytic complement (CH50), and complement components C3 and C4 were determined in 550 serum samples taken from patients with active (311 samples) or inactive (239) systemic lupus erythematosus (SLE). Increased anti-DNA levels were shown in 82% of the samples from patients with active and in 57.8% of those from inactive, disease. Decreased levels of CH50, and C3, C4 were found in 37, 50, and 80% of the samples taken from active and in 22, 29, and 67% of those from inactive, disease, respectively. Positive ANA test was found in 94.7% of the patients with active and 87.9% of those with inactive, disease. Significant differences were found between the two groups for all parameters. The correlations were close between the values of anti-DNA antibodies and CH50, as well as between the levels of CH50 and C3. Relationship between anti-DNA antibodies and C3 levels, as well as between CH50 and C4 levels was also demonstrated. Six subgroups for expressing the positivity of five parameters, 32 patterns of positive parameters were found and their possible application were suggested. In combining these parameters and using appropriate patterns, their determination may be helpful not only for the diagnosis but also for the assessment of disease activity.

[Effect of female sex hormones on blastic transformation of lymphocytes in systemic lupus erythematosus and in healthy women]. / A nöl nemi hormonok hatása a lymphocyta blastos transformatióra systhemás lupus erythematosusban és egészséges nökben.

PIP: The susceptibility of women to autoimmune diseases is well-documented, of which systemic lupus erythematosus (SLE) is especially important. The use of oral contraceptives often activate SLE from a quiescent condition. The inductive effect of estrogen has been shown in animal studies indicating that female hormones can trigger autoimmune reaction. The effect of ethinyl estradiol, an estrogen (E), and d-norgestrel, a progesterone (P), on the mitogenic response of peripheral lymphocytes, and particularly on phytohemagglutinin (PHA)- and concanavalin-A (Con-A)-induced blastic transformation of lymphocytes (LBT) was studied in vitro. 25 patients with SLE and 27 healthy controls participated in the study. SLE was inactive in 16 patients, 7 took corticosteroids, and 3 also received 50 mg/day Imuran. 13 patients and 10 controls took contraceptives (Bisecurin, Infecundin, Ovidon, Rigevidon). The LBT value fell significantly in patients with active SLE, in contraceptive users, and the value was significantly lower in inactive patients than in those not using contraceptives. E and P separately or together significantly reduced LBT values. Contraceptives containing P only can be prescribed for women suffering from SLE, as its role in inducing the disease compared to E is negligible.^ieng