Loss of the ability to generate large burst-forming unit-like megakaryocytic colonies from thawed cord blood in semisolid cultures after short term suspension culture.

BACKGROUND AND OBJECTIVES: Total colony-forming cells from thawed cord blood units (CBUs) include megakaryocytic colony-forming units (CFU-Mks), which survive the freezing process. The aim of this study was to evaluate whether different megakaryocytic progenitors from unseparated CBUs survive the freezing process and a short-term liquid culture. MATERIALS AND METHODS: Thawed samples of CBUs were cultured in liquid medium. During the cultures, serial samples were drawn to assess the growth of different megakaryocytic progenitors in a semisolid collagen medium with identical cytokines as in the liquid medium. Megakaryocytic cells were detected using immunohistochemistry and flow cytometry. RESULTS: In suspension culture, the megakaryocytic progenitors almost completely lost the ability to generate large (burst-forming unit-like, BFU-like) megakaryocytic colonies in semisolid cultures (large colonies, median count per chamber d0: 7.25 vs. d7: 1.5; P < 0.0001), whereas the number of small colonies (median count per chamber d0: 7.25 vs. d7: 16.0; P = 0.0505) peaked at day seven. Further 7-day culture in suspension resulted in the decline of small colonies as well (d7: 16.0 vs. d14: 5.75; P = 0.0088). Total CFU-Mk count declined from 23.3 (range 12.5-34.0) at d0 to 7.25 (range 1.0-13.5) at d14 (P < 0.0001). CONCLUSION: Immediately post-thaw, CBUs possess an ability to generate large BFU-like megakaryocytic colonies, whereas the colonies were not detectable in most CBUs in semisolid culture after a short suspension culture. Small CFU-Mks were observed throughout the cultures. It may be that the BFU-Mk colonies matured and acquired CFU-Mk behaviour.

Francisella tularensis subspecies holarctica occurs in Swedish mosquitoes, persists through the developmental stages of laboratory-infected mosquitoes and is transmissible during blood feeding.

In Sweden, mosquitoes are considered the major vectors of the bacterium Francisella tularensis subsp. holarctica, which causes tularaemia. The aim of this study was to investigate whether mosquitoes acquire the bacterium as aquatic larvae and transmit the disease as adults. Mosquitoes sampled in a Swedish area where tularaemia is endemic (Örebro) were positive for the presence of F. tularensis deoxyribonucleic acid throughout the summer. Presence of the clinically relevant F. tularensis subsp. holarctica was confirmed in 11 out of the 14 mosquito species sampled. Experiments performed using laboratory-reared Aedes aegypti confirmed that F. tularensis subsp. holarctica was transstadially maintained from orally infected larvae to adult mosquitoes and that 25% of the adults exposed as larvae were positive for the presence of F. tularensis-specific sequences for at least 2 weeks. In addition, we found that F. tularensis subsp. holarctica was transmitted to 58% of the adult mosquitoes feeding on diseased mice. In a small-scale in vivo transmission experiment with F. tularensis subsp. holarctica-positive adult mosquitoes and susceptible mice, none of the animals developed tularaemia. However, we confirmed that there was transmission of the bacterium to blood vials by mosquitoes that had been exposed to the bacterium in the larval stage. Taken together, these results provide evidence that mosquitoes play a role in disease transmission in part of Sweden where tularaemia recurs.

Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease.

Initially identified in high-grade gliomas, mutations in the PTEN tumor-suppressor are also found in many sporadic cancers and a few related autosomal dominant hamartoma syndromes. PTEN is a 3'-specific phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) phosphatase and functions as a negative regulator of PI3K signaling. We generated a tissue-specific deletion of the mouse homolog Pten to address its role in brain function. Mice homozygous for this deletion (PtenloxP/loxP;Gfap-cre), developed seizures and ataxia by 9 wk and died by 29 wk. Histological analysis showed brain enlargement in PtenloxP/loxP;Gfap-cre mice as a consequence of primary granule-cell dysplasia in the cerebellum and dentate gyrus. Pten mutant cells showed a cell-autonomous increase in soma size and elevated phosphorylation of Akt. These data represent the first evidence for the role of Pten and Akt in cell size regulation in mammals and provide an animal model for a human phakomatosis condition, Lhermitte-Duclos disease (LDD).

Critical involvement of the thalamus and precuneus during restoration of consciousness with physostigmine in humans during propofol anaesthesia: a positron emission tomography study.

BACKGROUND: Functional brain imaging offers a way to investigate how general anaesthetics impair consciousness. However, functional imaging changes may result from drug effects unrelated to hypnosis. Establishing a causal link with loss of consciousness is thus difficult. METHODS: To identify changes of neuronal activity functionally linked to the level of consciousness, physostigmine was used to restore consciousness without changing the anaesthetic concentration in 11 subjects anaesthetized with propofol. Eight subjects (responders) regained consciousness after physostigmine and three did not (non-responders). Positron emission tomography was used to measure regional cerebral blood flow (rCBF); during baseline (awake), after anaesthesia-induced loss of consciousness, after physostigmine administration, and recovery. In addition to subtraction analyses, we used conjunction analysis in the responders to identify changes common to the baseline-anaesthesia and physostigmine-anaesthesia contrasts. RESULTS: Complete data were available for seven subjects (four responders and three non-responders). The analyses revealed that unconsciousness was associated with rCBF decreases in the thalamus and precuneus. Restoration of consciousness by physostigmine was associated with rCBF increases in these same structures, with the strongest effect in the thalamus. CONCLUSIONS: The results provide strong evidence that reductions in rCBF in the thalamus and precuneus are functionally related to propofol-induced unconsciousness independently of any non-specific effects of propofol. These observations confirm that the thalamus and precuneus are key elements to understand how general anaesthetics cause unconsciousness and how patients wake up from anaesthesia. Furthermore, they are consistent with the notion that anaesthetic-induced unconsciousness is associated with reduced cholinergic activation.

Stimulus-induced EEG-patterns and outcome after cardiac arrest.

OBJECTIVE: EEG is commonly used to predict prognosis in post anoxic coma. We investigated if stimulus-induced rhythmic, periodic or ictal discharges (SIRPIDs) add prognostic information after cardiac arrest. METHODS: In the multicenter Targeted Temperature Management trial, routine-EEGs were prospectively recorded after rewarming (≥36 h). Presence and subtype of SIRPIDs and main EEG-pattern (benign, malignant, highly malignant) were retrospectively reported according to a standardised classification. Patients were followed up after 180 days. Poor outcome was defined as severe neurological disability or death (Cerebral Performance Category 3-5). RESULTS: Of 142 patients, 71% had poor outcome and 14% had SIRPIDs. There was no significant difference in outcome between patients with and without SIRPIDs, even when subgrouped according to underlying main EEG-pattern. Comparing subtypes of SIRPIDs, 82% of patients with stimulus-induced periodic discharges had poor outcome compared to 44% of patients with stimulus-induced rhythmic delta activity, but the difference was not significant. CONCLUSIONS: In EEGs performed ≥36 h after cardiac arrest, SIRPIDs cannot be used to reliably predict poor outcome. Whether certain subtypes of SIRPIDs indicate worse prognosis needs further investigation. SIGNIFICANCE: Categorising the main EEG-pattern has important prognostic implications, but assessment of late appearing SIRPIDs does not seem to add prognostic information.

Atomised lidocaine for airway topical anaesthesia in the morbidly obese: 1% compared with 2%.

Airway anaesthesia using atomised lidocaine for awake oral fibreoptic intubation in morbidly obese patients was evaluated using two doses of local anaesthetic. In this randomised, blinded prospective study, 40 ml of atomised 1% (n = 11) or 2% (n = 10) lidocaine was administered with high oxygen flow as carrier. Outcomes included time for intubation, patient tolerance to airway manipulation, haemodynamic parameters, the bronchoscopist's overall satisfaction, and serial serum lidocaine concentrations. Patients receiving lidocaine 1% had a longer mean (SD) time from the start of topicalisation to tracheal tube cuff inflation than those receiving lidocaine 2% (8.6 (0.9) min vs 6.9 (0.5) min, respectively; p < 0.05). Patients in the 1% cohort demonstrated increased responses to airway manipulation (p < 0.0001), reflecting lower bronchoscopist's satisfaction scores (p < 0.03). Haemodynamic responses to topicalisation and airway manipulation were similar in both groups. Peak plasma concentration was lower in the 1% group (mean (SD) 1.4 (0.3) and 3.8 (0.5) microg.ml(-1), respectively; p < 0.001). Airway anaesthesia using atomised lidocaine for awake oral fibreoptic intubation in the morbidly obese is efficacious, rapid and safe. Compared with lidocaine 1%, the 2% dose provides superior intubating conditions.

Stratifying comatose postanoxic patients for somatosensory evoked potentials using routine EEG.

INTRODUCTION: Multimodal neurological prognostication is recommended for comatose patients after cardiac arrest. The absence of cortical N20-potentials in a somatosensory evoked potential (SSEP) examination reliably predicts poor outcome, but presence of N20-potentials have limited prognostic value. A benign routine electroencephalogram (EEG) may identify patients with a favourable prognosis who are likely to have present N20-potentials. OBJECTIVE: To investigate whether a routine EEG can identify patients where SSEP is unnecessary to perform. METHODS: In a multi-centre trial, comatose patients after cardiac arrest were randomised to a controlled temperature of 33 °C or 36 °C. A routine EEG was protocolised and SSEP performed at the clinicians' discretion, both during normothermic conditions. EEGs were categorised into benign, malignant or highly malignant based on standardised terminology. A benign EEG was defined as a continuous normal-voltage background without abundant discharges. The N20-potentials were reported as absent (bilaterally) or present (bilaterally or unilaterally). RESULTS: Both EEG and SSEP were performed in 161 patients. EEG was performed before SSEP in 60%. A benign EEG was seen in 29 patients and 100% (CI 88-100%) had present N20-potentials. For the 69 patients with a malignant EEG and the 63 patients with a highly malignant EEG, 67% (CI 55-77%) and 44% (CI 33-57%) had present N20-potentials, respectively. CONCLUSIONS: All patients with a benign EEG had present N20-potentials, suggesting that SSEP may be omitted in these patients to save resources. SSEP is useful in patients with a malignant or highly malignant EEG since these patterns are associated with both present and absent N20-potentials.

Predators and nutrient availability favor protozoa-resisting bacteria in aquatic systems.

The long co-existence of bacteria and protozoa has led to the development of bacterial protozoa resistance strategies, which are suggested to serve as drivers for the evolution of pathogenic bacteria. However, the ecological mechanisms underpinning selection for protozoa-resistance in aquatic bacteria are poorly known. To assess the role of nutrient availability and predation-pressure on selection for protozoa-resisting bacteria (PRB), an enrichment-dilution experiment was designed using laboratory microcosms containing natural lake water. PRB was monitored by screening 16S rRNA amplicon sequence data for reads assigned to bacteria that previously has been shown to resist degradation by amoebae. To estimate the effects of the microbial food web dynamics (microscopy of; heterotrophic bacteria, phytoplankton, protozoa and rotifers) and physicochemical variables on the PRB abundance in the study system, a joint species distribution modelling approach was used. The predation-pressure (ratio between predator and bacterial biomass) had a positive effect on the abundance of the PRB genus Mycobacterium, while perturbation (enrichment and dilution) favored the PRB genus Pseudomonas that dominated the bacterial community in the disturbed systems. Our results show that PRB with different ecological strategies can be expected in water of high and intermediate nutrient levels and after major disturbances of an aquatic system.

Highly malignant routine EEG predicts poor prognosis after cardiac arrest in the Target Temperature Management trial.

INTRODUCTION: Routine EEG is widely used and accessible for post arrest neuroprognostication. Recent studies, using standardised EEG terminology, have proposed highly malignant EEG patterns with promising predictive ability. OBJECTIVES: To validate the performance of standardised routine EEG patterns to predict neurological outcome after cardiac arrest. METHODS: In the prospective multicenter Target Temperature Management trial, comatose cardiac arrest patients were randomised to different temperature levels (950 patients, 36 sites). According to the prospective protocol a routine EEG was performed in patients who remained comatose after the 36 h temperature control intervention. EEGs were retrospectively reviewed blinded to outcome using the standardised American Clinical Neurophysiology Society terminology. Highly malignant, malignant and benign EEG patterns were correlated to poor and good outcome, defined by best achieved Cerebral Performance Category up to 180 days. RESULTS: At 20 sites 207 patients had a routine EEG performed at median 76 h after cardiac arrest. Highly malignant patterns (suppression or burst-suppression with or without discharges) had a high specificity for poor outcome (98%, CI 92-100), but with limited sensitivity (31%, CI 24-39). Our false positive patient had a burst-suppression pattern during ongoing sedation. A benign EEG, i.e. continuous normal-voltage background without malignant features, identified patients with good outcome with 77% (CI 66-86) sensitivity and 80% (CI 73-86) specificity. CONCLUSION: Highly malignant routine EEG after targeted temperature management is a strong predictor of poor outcome. A benign EEG is an important indicator of a good outcome for patients remaining in coma.

Effects of GABA and glycine on sympathetic preganglionic neurons in the upper thoracic intermediolateral nucleus of the cat.

GABA (5-137 nA) and glycine (5-75 nA) each inhibit spontaneous activity and block antidromic invasion of the soma-dendritic region of single sympathetic preganglionic neurons (SPNs) in the intermediolateral nucleus of T1-T3 in the cat. These effects are rapid in onset and recovery. They are selectively blocked by bicuculline and strychnine respectively. Thus, GABA and glycine exert pharmacologically specific inhibitory effects on SPNs and this supports the possibility that they may be chemical mediators of inhibitory inputs directly onto these neurons.

Effects of glutamate and aspartate on sympathetic preganglionic neurons in the upper thoracic intermediolateral nucleus of the cat.

Glutamate and aspartate excited all spontaneously active sympathetic preganglionic neurons (SPNs) tested in the intermediolateral nucleus of spinal segments T1-T3. Most silent neurons could be induced to discharge but the remainder showed only a decrease in antidromic spike amplitude. These effects were typically fast (on, off less than 1 s). D,L-Homocysteic acid also produced excitation; this effect was typically slower. Glutamate and aspartate were usually equipotent; 20% were differentially sensitive to aspartate, 10% to glutamate.

Effects of oxytocin and vasopressin on thoracic sympathetic preganglionic neurones in the cat.

When applied by iontophoresis onto single sympathetic preganglionic neurones in the intermediolateral nucleus of segments T1-T3 in the cat, oxytocin and vasopressin each had an excitatory effect. This effect consisted of a prolonged (30-300 sec) after-discharge following termination of application. These results indicate that oxytocin and vasopressin each exert excitatory effects on sympathetic preganglionic neurones and support the possibility that they may be chemical mediators of synaptic transmission in the intermediolateral nucleus, perhaps in cardioacceleratory and/or pressor pathways descending from the paraventricular nucleus of the hypothalamus.

The role of excess subcutaneous fat in pain and sensory sensitivity in obesity.

BACKGROUND: Previous studies suggest pain sensitivity may be decreased in obesity, but it is unknown whether this is a global or a site-specific phenomenon related to the amount of excess fat. DESIGN: a cross-sectional study comparing obese and non-obese participants on body sites with much and little excess subcutaneous fat in obesity. Hot and cold sensory detection thresholds, pain thresholds, pain tolerance and subjective ratings for a cold (0 °C) and hot (48 °C) stimulus were assessed using a 16 × 16 mm thermode (Medoc, Israel) on the forehead and abdomen. Pressure pain thresholds were measured on the hand. Cold water immersion tolerance duration and subjective ratings were assessed on the hand. Two indices of central pain processing, i.e., temporal summation and heterotopic noxious stimulation, were assessed. RESULTS: A total of 20 obese participants [10M/10F, BMI mean (SD) =41.5 kg/m(2) (9.4 kg/m(2) )] and 20 age- and gender-matched non-obese controls [10M/10F, BMI mean (SD) =23.5 kg/m(2) (2.9 kg/m(2) )] were studied. Compared with non-obese, obese participants had higher thresholds and lower subjective ratings, indexing decreased sensitivity, for painful and non-painful thermal stimuli on the abdomen, an area with much excess subcutaneous fat. Decreases in abdominal sensitivity correlated with measures of adiposity (i.e., waist-to-hip ratio and subcutaneous fat thickness). On areas with little excess subcutaneous fat (forehead and hand), obese and non-obese groups did not differ in measures of thermal or pressure sensitivity, nor for indices of central pain processing. CONCLUSION: Obese participants are less sensitive than non-obese individuals, but only on areas with excess subcutaneous fat.

Molecular Detection of Persistent Francisella tularensis Subspecies holarctica in Natural Waters.

Tularemia, caused by the bacterium Francisella tularensis, where F. tularensis subspecies holarctica has long been the cause of endemic disease in parts of northern Sweden. Despite this, our understanding of the natural life-cycle of the organism is still limited. During three years, we collected surface water samples (n = 341) and sediment samples (n = 245) in two areas in Sweden with endemic tularemia. Real-time PCR screening demonstrated the presence of F. tularenis lpnA sequences in 108 (32%) and 48 (20%) of the samples, respectively. The 16S rRNA sequences from those samples all grouped to the species F. tularensis. Analysis of the FtM19InDel region of lpnA-positive samples from selected sampling points confirmed the presence of F. tularensis subspecies holarctica-specific sequences. These sequences were detected in water sampled during both outbreak and nonoutbreak years. Our results indicate that diverse F. tularensis-like organisms, including F. tularensis subsp. holarctica, persist in natural waters and sediments in the investigated areas with endemic tularemia.