Twelve years' detection of respiratory viruses by immunofluorescence in hospitalised children: impact of the introduction of a new respiratory picornavirus assay

Background Direct immunofluorescence assays (DFA) are a rapid and inexpensive method for the detection of respiratory viruses and may therefore be used for surveillance. Few epidemiological studies have been published based solely on DFA and none included respiratory picornaviruses and human metapneumovirus (hMPV). We wished to evaluate the use of DFA for epidemiological studies with a long-term observation of respiratory viruses that includes both respiratory picornaviruses and hMPV. Methods Since 1998 all children hospitalized with respiratory illness at the University Hospital Bern have been screened with DFA for common respiratory viruses including adenovirus, respiratory syncytial virus (RSV), influenza A and B, and parainfluenza virus 1-3. In 2006 assays for respiratory picornaviruses and hMPV were added. Here we describe the epidemiological pattern for these respiratory viruses detected by DFA in 10'629 nasopharyngeal aspirates collected from 8'285 patients during a 12-year period (1998-2010). Results Addition of assays for respiratory picornaviruses and hMPV raised the proportion of positive DFA results from 35% to 58% (p < 0.0001). Respiratory picornaviruses were the most common viruses detected among patients ≥1 year old. The seasonal patterns and age distribution for the studied viruses agreed well with those reported in the literature. In 2010, an hMPV epidemic of unexpected size was observed. Conclusions DFA is a valid, rapid, flexible and inexpensive method. The addition of assays for respiratory picornaviruses and hMPV broadens its range of viral detection. DFA is, even in the "PCR era", a particularly adapted method for the long term surveillance of respiratory viruses in a pediatric population.

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5% v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS² results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2 μg/mL (extracts).

Comparison among gold standard techniques used for the validation of methods for occlusal caries detection

The aim of this in vitro study was to assess the agreement among four techniques used as gold standard for the validation of methods for occlusal caries detection. Sixty-five human permanent molars were selected and one site in each occlusal surface was chosen as the test site. The teeth were cut and prepared according to each technique: stereomicroscopy without coloring (1), dye enhancement with rhodamine B (2) and fuchsine/acetic light green (3), and semi-quantitative microradiography (4). Digital photographs from each prepared tooth were assessed by three examiners for caries extension. Weighted kappa, as well as Friedman's test with multiple comparisons, was performed to compare all techniques and verify statistical significant differences. Results: kappa values varied from 0.62 to 0.78, the latter being found by both dye enhancement methods. Friedman's test showed statistical significant difference (P < 0.001) and multiple comparison identified these differences among all techniques, except between both dye enhancement methods (rhodamine B and fuchsine/acetic light green). Cross-tabulation showed that the stereomicroscopy overscored the lesions. Both dye enhancement methods showed a good agreement, while stereomicroscopy overscored the lesions. Furthermore, the outcome of caries diagnostic tests may be influenced by the validation method applied. Dye enhancement methods seem to be reliable as gold standard methods.

Magnetization exchange observed in human skeletal muscle by non-water-suppressed proton magnetic resonance spectroscopy

Many metabolites in the proton magnetic resonance spectrum undergo magnetization exchange with water, such as those in the downfield region (6.0-8.5 ppm) and the upfield peaks of creatine, which can be measured to reveal additional information about the molecular environment. In addition, these resonances are attenuated by conventional water suppression techniques complicating detection and quantification. To characterize these metabolites in human skeletal muscle in vivo at 3 T, metabolite cycled non-water-suppressed spectroscopy was used to conduct a water inversion transfer experiment in both the soleus and tibialis anterior muscles. Resulting median exchange-independent T(1) times for the creatine methylene resonances were 1.26 and 1.15 s, and for the methyl resonances were 1.57 and 1.74 s, for soleus and tibialis anterior muscles, respectively. Magnetization transfer rates from water to the creatine methylene resonances were 0.56 and 0.28 s(-1) , and for the methyl resonances were 0.39 and 0.30 s(-1) , with the soleus exhibiting faster transfer rates for both resonances, allowing speculation about possible influences of either muscle fibre orientation or muscle composition on the magnetization transfer process. These water magnetization transfer rates observed without water suppression are in good agreement with earlier reports that used either postexcitation water suppression in rats, or short CHESS sequences in human brain and skeletal muscle.

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.

Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA

PURPOSE: To investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, (177)Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3. METHODS: In vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand (177)Lu-AMBA was used and compared with established bombesin radioligands such as (125)I-Tyr(4)-bombesin and (125)I-[D: -Tyr(6),beta-Ala(11),Phe(13),Nle(14)]-bombesin(6-14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour. RESULTS: (177)Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. (177)Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with (177)Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with (177)Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with (177)Lu-AMBA than with the conventional bombesin radioligands. CONCLUSION: The present in vitro study suggests that (177)Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessels.

Influence of the condition of the adjacent tooth surface on fluorescence measurements for the detection of approximal caries

The aim of this study was to test whether the status of the adjacent tooth surface has an influence on the signal of a new laser fluorescence (LF) device for the detection of approximal caries. Seventy-eight teeth were selected from a pool of extracted permanent human molars, frozen at -20 degrees C until use. Before being measured the teeth were defrosted, cleaned, and any calculus removed. As a control, a defined approximal surface of each tooth was measured with the LF device holding the tip with the detecting- and the reverse-side on it, but without a neighboring tooth contacting the surface. The proximal site under examination was then placed adjacent to a tooth, which had deep dentinal caries, a composite restoration, a provisional ZnO-Eugenol restoration, or a ceramic restoration. The adjacent tooth with the ZnO-Eugenol restoration, the composite restoration, and the dentinal caries all demonstrated a statistically significant increase of LF readings on sound tooth surfaces. Teeth with enamel or dentinal caries were only slightly (and not statistically significantly) influenced by the different types of neighboring surfaces compared with the control LF readings. It can be concluded that caries detection of approximal tooth surfaces with the new LF system might be influenced by the condition of the adjacent tooth surface.

Dissociated lateralization of transient and sustained blood oxygen level-dependent signal components in human primary auditory cortex

Among other auditory operations, the analysis of different sound levels received at both ears is fundamental for the localization of a sound source. These so-called interaural level differences, in animals, are coded by excitatory-inhibitory neurons yielding asymmetric hemispheric activity patterns with acoustic stimuli having maximal interaural level differences. In human auditory cortex, the temporal blood oxygen level-dependent (BOLD) response to auditory inputs, as measured by functional magnetic resonance imaging (fMRI), consists of at least two independent components: an initial transient and a subsequent sustained signal, which, on a different time scale, are consistent with electrophysiological human and animal response patterns. However, their specific functional role remains unclear. Animal studies suggest these temporal components being based on different neural networks and having specific roles in representing the external acoustic environment. Here we hypothesized that the transient and sustained response constituents are differentially involved in coding interaural level differences and therefore play different roles in spatial information processing. Healthy subjects underwent monaural and binaural acoustic stimulation and BOLD responses were measured using high signal-to-noise-ratio fMRI. In the anatomically segmented Heschl's gyrus the transient response was bilaterally balanced, independent of the side of stimulation, while in opposite the sustained response was contralateralized. This dissociation suggests a differential role at these two independent temporal response components, with an initial bilateral transient signal subserving rapid sound detection and a subsequent lateralized sustained signal subserving detailed sound characterization.

[(99m)Tc]Demotate 2 in the detection of sst(2)-positive tumours: a preclinical comparison with [(111)In]DOTA-tate

PURPOSE: The aim of this study was to evaluate [(99m)Tc]Demotate 2 ([(99m)Tc-N(4) (0-1),Asp(0),Tyr(3)]octreotate) as a candidate for in vivo imaging of sst(2)-positive tumours and to compare it with [(111)In]DOTA-tate ([(111)In-DOTA(0),Tyr(3)]octreotate). METHODS: Labelling of Demotate 2 with (99m)Tc was performed at room temperature using SnCl(2) as reductant in the presence of citrate at alkaline pH. Radiochemical analysis involved ITLC and HPLC methods. Peptide conjugate affinities for sst(2) were determined by receptor autoradiography on rat brain cortex sections using [DOTA(0),(125)I-Tyr(3)]octreotate as the radioligand. The affinity profile of Demotate 2 for human sst(1)-sst(5) was studied by receptor autoradiography in cell preparations using the universal somatostatin radioligand [(125)I][Leu(8),(D: )Trp(22),Tyr(25)]somatostatin-28. The internalisation rates of [(99m)Tc]Demotate 2 and [(111)In]DOTA-tate were compared in sst(2)-positive and -negative control cell lines. Biodistribution of radiopeptides was studied in male Lewis rats bearing CA20948 tumours. RESULTS: Peptide conjugates showed selectivity and a high affinity binding for sst(2) (Demotate 2 IC(50)=3.2 nM and DOTA-tate IC(50)=5.4 nM). [(99m)Tc]Demotate 2, like [(111)In]DOTA-tate, internalised rapidly in all sst(2)-positive cells tested, but not in sst(2)-negative control cells. After injection in CA20948 tumour-bearing rats both radiopeptides showed high and specific uptake in the sst(2)-positive organs and in the implanted tumour and rapid excretion from non-target tissues via the kidneys. CONCLUSION: [(99m)Tc]Demotate 2, similarly to the known sst(2)-targeting agent [(111)In]DOTA-tate, showed promising biological qualities for application in the scintigraphy of sst(2)-positive tumours.

Tracking the subprocesses of decision-based action in the human frontal lobes

Situationally adaptive behavior relies on the identification of relevant target stimuli, the evaluation of these with respect to the current context and the selection of an appropriate action. We used functional magnetic resonance imaging (fMRI) to disentangle the neural networks underlying these processes within a single task. Our results show that activation of mid-ventrolateral prefrontal cortex (PFC) reflects the perceived presence of a target stimulus regardless of context, whereas context-appropriate evaluation is subserved by mid-dorsolateral PFC. Enhancing demands on response selection by means of response conflict activated a network of regions, all of which are directly connected to motor areas. On the midline, rostral anterior paracingulate cortex was found to link target detection and response selection by monitoring for the presence of behaviorally significant conditions. In summary, we provide new evidence for process-specific functional dissociations in the frontal lobes. In target-centered processing, target detection in the VLPFC is separable from contextual evaluation in the DLPFC. Response-centered processing in motor-associated regions occurs partly in parallel to these processes, which may enhance behavioral efficiency, but it may also lead to reaction time increases when an irrelevant response tendency is elicited.

Detection of approximal caries with a new laser fluorescence device

The laser device DIAGNOdent developed for the detection of occlusal caries has limited value on approximal surfaces. The aim of this study was to develop and to test a new laser fluorescence (LF) device for the detection of approximal caries. Light with a wavelength of 655 nm was transported to the approximal surface using two different sapphire fibre tips. Seventy-five teeth were selected from a pool of extracted permanent human molars, frozen at -20 degrees C until use. Before being measured, they were defrosted, cleaned and calculus was removed with a scaler. The molars were set in blocks simulating the contact area of adults. Bitewing radiographs were obtained using Kodak Insight films. After two independent assessments with the new LF device, the teeth were histologically prepared, and assessed for caries extension. Using the laser, specificity values for D1 threshold (outer half of enamel), D2 threshold (inner half of enamel), D3 threshold (dentine) ranged between 0.81 and 0.93, sensitivity between 0.84 and 0.92 with no difference between the two tips. Bitewing radiography showed an inferior performance compared to LF (p<0.05). Intraex aminer reproducibility was high (kappa>.74). The new LF system might be a useful additional tool in detecting approximal caries. Because of its good reproducibility, it could be used to monitor caries regression or progression on approximal surfaces.

Performance of a new laser fluorescence device for the detection of occlusal caries in vitro

The new device DIAGNOdent pen based on red laser light induced fluorescence was introduced for the detection of approximal and occlusal caries. The aim of this study was to test its performance on occlusal surfaces. The new device comes with two different sapphire fibre tips: a cylindrical tip and a conical tip. The two new sapphire fibre tips were used and compared with the tip currently available with DIAGNOdent (DD). METHODS: The teeth were selected from a pool of extracted permanent human molars, which were stored frozen at -20 degrees C, until use. Prior to being measured the teeth were defrosted and cleaned. One hundred and nineteen teeth were selected and measured with the old tip and with the two new tips of the new device by two independent assessments. The teeth were histologically prepared and assessed for caries extension. RESULTS: Specificity values for D(1), D(2) and D(3) ranged between 0.69 and 0.89, sensitivity between 0.78 and 0.96. There were no statistically significant differences obtained between the two tips of the new and the one tip of the old device (p>0.05). Intra-examiner reliability with kappa values of >0.83 was high. CONCLUSIONS: In this study, the new laser fluorescence device performed on occlusal surfaces as well as the available device.

Immunodetection of bone-related proteins in microcalcifications of human dental pulp

Objective: Root canal obliterations may pose esthetic and clinical problems or may even be a risk factor for tooth survival. Microcalcifications in the pulp can be so extensive that the entire root canal system becomes obliterated. Since bone sialoprotein (BSP) and osteopontin (OPN) are involved in both physiological and pathological mineralization processes, our hypothesis was that these two bone-related noncollagenous proteins are present in microcalcifications of the pulp. The purpose of this study was, therefore, to characterize the nature of microcalcifications in the pulp of aged human teeth. Methods: From a large collection of human teeth, 10 were found to exhibit pulpal microcalcifications. The teeth were extracted for periodontal reasons from 39-60 year old patients. After fixation in aldehydes and decalcification, teeth were processed for embedding in LR White resin for analysis in the light and transmission electron microscope. For the detection of BSP and OPN, post-embedding high resolution immunocytochemistry was applied. Results: The microcalcifications were round or elongated, occasionally coalescing, and intensely stained with toluidine blue. Collagen fibrils were found in most but not all microcalcifications. All microcalcifications were immunoreactive for both antibodies and showed an identical labeling pattern. Gold particle labeling was extensively found throughout the interfibrillar ground substance of the microcalcifications, whereas the dentin matrix lacked immunolabeling. Conclusion: BSP and OPN appear to be major matrix constituents of pulp microcalcifications and may thus, like in other mineralized tissues, be involved in their mineralization process.

Human group II 14 kDa phospholipase A2 activates human platelets

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

Stimulation-specific contribution of p38 and JNK to IFN-beta gene expression in human macrophages

Induction of interferon-beta (IFN-beta) gene expression is a tightly regulated process, and a plethora of studies identified the signal transduction pathway TANK-binding kinase-1 (TBK-1)/IFN regulatory factor-3 (IRF-3) as essential to the induction of IFN-beta gene expression. Data regarding the role of p38 and JNK are rare, however. We investigated the contribution of these kinases to IFN-beta expression in human macrophages treated with poly(I:C), lipopolysaccharide (LPS), Sendai virus, or vesicular stomatitis virus (VSV). We found that all the stimuli induced IFN-beta mRNA, albeit to a different extent. Whereas LPS and VSV induced the phosphorylation of p38 and JNK, neither poly(I:C) nor Sendai virus led to the detection of phosphospecific signals. When inhibiting p38, a VSV-triggered IFN-beta mRNA response was inhibited, whereas inhibiting JNK suppressed an LPS-triggered response, but only when macrophages were primed with IFN-gamma. Neither poly(I:C)-induced nor Sendai virus-induced IFN-beta mRNA expression was affected when p38 and JNK were inhibited. Collectively, the data show that the contribution of p38 and JNK to the expression of IFN-beta occurs in a stimulation-specific manner in human macrophages.