Respiratory viral infections are underdiagnosed in patients with suspected sepsis.

The study aim was to investigate the prevalence and clinical relevance of viral findings by multiplex PCR from the nasopharynx of clinically septic patients during a winter season. During 11 weeks of the influenza epidemic period in January-March 2012, consecutive adult patients suspected to be septic (n = 432) were analyzed with cultures from blood and nasopharynx plus multiplex PCR for respiratory viruses on the nasopharyngeal specimen. The results were compared with those from microbiology analyses ordered as part of standard care. During the winter season, viral respiratory pathogens, mainly influenza A virus, human metapneumovirus, coronavirus, and respiratory syncytial virus were clinically underdiagnosed in 70% of patients positive by the multiplex PCR assay. During the first four weeks of the influenza epidemic, few tests for influenza were ordered by clinicians, indicating low awareness that the epidemic had started. Nasopharyngeal findings of Streptococcus pneumoniae and Haemophilus influenzae by culture correlated to pneumonia diagnosis, and in those patients laboratory signs of viral co-infections were common but rarely suspected by clinicians. The role of respiratory viral infections in patients presenting with a clinical picture of sepsis is underestimated. Specific antiviral treatment might be beneficial in some cases and may reduce spread in a hospital setting. Diagnosing viral infections may promote reduction of unnecessary antibiotic use. It can also be a tool for decisions concerning patient logistics, in order to minimize exposure of susceptible patients and personnel.

Case fatality ratio and mortality rate trends of community-onset Staphylococcus aureus bacteraemia.

Lethal outcomes can be expressed as a case fatality ratio (CFR) or as a mortality rate per 100 000 population per year (MR). Population surveillance for community-onset methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus bacteraemia was conducted in Canada, Australia, Sweden and Denmark to evaluate 30-day CFR and MR trends between 2000 and 2008. The CFR was 20.3% (MSSA 20.2%, MRSA 22.3%) and MR was 3.4 (MSSA 3.1, MRSA 0.3) per 100 000 per year. Although MSSA CFR was stable the MSSA MR increased; MRSA CFR decreased while its MR remained low during the study. Community-onset S. aureus bacteraemia, particularly MSSA, is associated with major disease burden. This study highlights complementary information provided by evaluating both CFR and MR.

The changing epidemiology of Staphylococcus aureus bloodstream infection: a multinational population-based surveillance study.

Although the epidemiology of Staphylococcus aureus bloodstream infection (BSI) has been changing, international comparisons are lacking. We sought to determine the incidence of S. aureus BSI and assess trends over time and by region. Population-based surveillance was conducted nationally in Finland and regionally in Canberra, Australia, western Sweden, and three areas in each of Canada and Denmark during 2000-2008. Incidence rates were age-standardized and gender-standardized to the EU 27-country 2007 population. During 83 million person-years of surveillance, 18,430 episodes of S. aureus BSI were identified. The overall annual incidence rate for S. aureus BSI was 26.1 per 100,000 population, and those for methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) were 24.2 and 1.9 per 100,000, respectively. Although the overall incidence of community-onset MSSA BSI (15.0 per 100,000) was relatively similar across regions, the incidence rates of hospital-onset MSSA (9.2 per 100,000), community-onset MRSA (1.0 per 100,000) and hospital-onset MRSA (0.8 per 100,000) BSI varied substantially. Whereas the overall incidence of S. aureus BSI did not increase over the study period, there was an increase in the incidence of MRSA BSI. Major changes in the occurrence of community-onset and hospital-onset MSSA and MRSA BSI occurred, but these varied significantly among regions, even within the same country. Although major changes in the epidemiology of community-onset and hospital-onset MSSA and MRSA BSIs are occurring, this multinational population-based study did not find that the overall incidence of S. aureus BSI is increasing.

Antibody responses in patients with invasive Staphylococcus aureus infections.

Correlation between antibody response and clinical outcome in Staphylococcus aureus bacteremia has yielded conflicting results. Immunization schedules have failed in clinical trials. Is the humoral response toward S. aureus of protective nature? A prospective study was performed in patients with invasive S. aureus (ISA) infections during the period 2003-2005. The antibody levels were determined at the beginning and at the end of treatment and one month later (n = 96, n = 71, and n = 51, respectively). As controls, 115 healthy individuals were used. A quantitative enzyme-linked immunosorbent assay (ELISA) against eight purified antigens was performed. Bacterial isolates were grouped as to the production of alpha-toxin, agr type, and pulsed-field gel electrophoresis (PFGE) type. Large variations were seen in the antibody levels. The levels in the second sample were the highest. A correlation between agr group, PFGE group, alpha-toxin production, and initial antibody levels was observed. Patients with fatal outcome displayed lower initial antibody levels to all antigens and significantly so in regard to teichoic acid, lipase, enterotoxin A, and scalded skin syndrome toxin. In episodes with complicated bacteremia, initial significantly low levels to teichoic acid and lipase were registered. Low initial antibody levels against several antigens were associated with increased mortality and complicated bacteremia in invasive S. aureus infections. Bacterial properties, strain, and toxin production affected the antibody response.

Outcome for invasive Staphylococcus aureus infections.

We report a survey of invasive Staphylococcus aureus (ISA) infections concerning outcome variables such as mortality, recurrence and residual symptoms. A prospective, population-based study of all cases of ISA was conducted in the catchment area of Skaraborg Hospital (population 255,109) in western Sweden during the period from 1st March 2003 to 28th February 2005. One hundred and fifty-seven patients were included. Recurrences were seen in 13 cases (9.3%). Thirty patients (19.1%) died during the first 28 days. Mortality rates for complicated bacteraemia and severe sepsis were 32% and 54%, respectively. Older patients (>65 years of age), patients with concomitant heart disease and patients with endovascular infections all suffered higher mortality. Line-associated infections had a higher recurrence rate. Residual symptoms were common, with 34% of the living patients reporting incomplete recovery. Accessory gene regulator (agr) type within the bacteria did not affect disease presentation. We conclude that ISA infections are of major medical importance, with high rates of mortality (19.1%), recurrence (9.3%) and residual functional impairment (34%).

Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms.

Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase. Today glutaredoxins are known as a multifunctional family of GSH-disulfide-oxidoreductases belonging to the thioredoxin fold superfamily. In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. The human Grx2 gene, located at chromosome 1q31.2--31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms that differed in their 5' region were identified. These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively. The 125-residue Grx domain and the two full-length variants were expressed in E. coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria. We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal.

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells.

The thiol antioxidant N-acetyl- L-cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL-2 and up-regulation of the IL-2 receptor. The 120-kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen-specific T cell clones (TCC). TCC were stimulated with anti-CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down-regulation of IL-4, IL-5 and IFN-gamma levels in Th0 and Th2 clones, with the most pronounced decrease of IL-4. Furthermore, they down-regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 m M NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down-regulation of IL-4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL-4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases.

Differential expression of SNAP-25a and SNAP-25b RNA transcripts in cranial nerve nuclei.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in the molecular regulation of neurotransmitter release. SNAP-25 exists in two isoforms, SNAP-25a and SNAP-25b, which arise from alternate splicing and which are differentially expressed throughout the nervous system. In situ hybridization was used to examine the presence and subcellular localization of SNAP-25a and SNAP-25b RNA transcript expression in motor and parasympathetic nuclei associated with cranial nerves. SNAP-25a RNA transcripts were strongly expressed in the parasympathetic Edinger-Westphal nucleus and dorsal motor nucleus of the vagus nerve but weakly expressed in motor nuclei such as the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal and accessory nuclei and in motoneurons of mouse lumbar spinal cord. In contrast, SNAP-25b RNA transcripts were not detectable in the Edinger-Westphal nucleus and dorsal motor nucleus of the vagus nerve but were strongly expressed in the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal, and accessory nuclei and in the motoneurons of mouse lumbar spinal cord. In the parasympathetic cranial nerve nuclei, displaying high levels of SNAP-25a RNA transcripts, the labeling was cytoplasmic, whereas the labeling was nuclear in the cranial nerve motor nuclei, displaying lower levels of transcripts. In contrast, labeling of SNAP-25b RNA transcripts was cytoplasmic in cranial nerve motor nuclei and not detectable in parasympathetic cranial nerve nuclei. Possible explanations for the region-specific and differential subcellular localization of SNAP-25a and SNAP-25b RNA transcripts are discussed.

Isoform-specific exocytotic protein mRNA expression in hypothalamic magnocellular neurons: regulation after osmotic challenge.

Exocytosis is regulated by proteins which interact to promote docking and fusion of secretory granules with the plasma membrane. We have used in situ hybridization to study the mRNA expression for vesicle-associated membrane protein (VAMP) isoforms VAMP-1 and VAMP-2, synaptosomal-associated protein of 25-kDa (SNAP-25) isoforms SNAP-25a and SNAP-25b, mammalian homologue of unc-18 (munc-18) and Hrs-2 in neurosecretory neurons of the magnocellular paraventricular (PVN) and supraoptic (SON) nuclei of normal and osmotically challenged animals. In PVN and SON neurons of normal animals, strong labeling was demonstrated for VAMP-2 and SNAP-25a mRNA, whereas VAMP-1 or SNAP-25b mRNA could not be detected. Salt-loading (2% NaCl as drinking water), an animal model which increases the expression and secretion of hormones from hypothalamic magnocellular neurons, resulted in significantly increased mRNA levels for VAMP-2 (36%, 28%), munc-18 (74%, 68%) and SNAP-25a (59%, 77%) in the PVN and SON, respectively. There was no significant increase in Hrs-2 mRNA levels in the PVN, whereas a significant increase (22%) was observed in the SON. In the posterior pituitary, immunohistochemistry showed a marked decrease in numbers and intensity of vasopressin-immunoreactive (-IR) nerve endings after salt-loading. There were no obvious changes in numbers or intensity of VAMP-2-, munc-18-, Hrs-2- or SNAP-25-IR fibers. Large varicosities containing VAMP-2- and Hrs-2 immunocreactivity were seen in salt-loaded animals. The results show isoform-specific mRNA expression in neurosecretory neurons and an increased mRNA expression of proteins participating in the molecular regulation of exocytosis during an experimental situation characterized by increased secretion.

VAMP-1 and VAMP-2 gene expression in rat spinal motoneurones: differential regulation after neuronal injury.

Vesicle-associated membrane protein (VAMP; synaptobrevin) is involved in the molecular regulation of transmitter release at the presynaptic plasma membrane. VAMP exists in two isoforms, VAMP-1 and VAMP-2, which are transcribed from two separate genes and differentially expressed in the nervous system. In situ hybridization was used to examine whether VAMP isoform mRNA expression may be altered by experimental manipulations. The effect of nerve injury on VAMP-1 and VAMP-2 mRNA levels in motoneurones of the rat lumbar spinal cord was compared with lesion-induced changes in the expression of choline acetyl transferase (ChAT) and alpha-calcitonin gene-related peptide (alpha-CGRP) mRNA. After unilateral sciatic nerve transection (axotomy), VAMP-1 mRNA expression decreased significantly in parallel with a downregulation of ChAT mRNA in axotomized motoneurones compared with the corresponding motoneurones on the contralateral unlesioned side. There was a rapid decrease in VAMP-1 and ChAT mRNA levels at 2 days after axotomy, and at 7 days there was a 65% decrease in VAMP-1 mRNA and a 48% decrease in ChAT mRNA. VAMP-1 mRNA levels continued to decrease at 14 and 21 days, while ChAT mRNA levels had returned to normal at this time. In contrast, VAMP-2 and alpha-CGRP mRNA levels were upregulated in axotomized motoneurones. A significant increase for both VAMP-2 and alpha-CGRP mRNA levels was present 2 days after axotomy, and a maximum was reached after 7 days for alpha-CGRP mRNA (163%) and after 14 days for VAMP-2 mRNA (587%). Immunohistochemical analysis did not reveal any detectable changes in VAMP-1- or VAMP-2-like immunoreactivity in the motoneurone cell soma after axotomy. In the proximal end of the transected sciatic nerve, there was an increase in VAMP-1- and VAMP-2-LI, which was most prominent at 2 days after lesion. The results show that, in axotomized spinal motoneurones, VAMP-1 mRNA is downregulated and VAMP-2 mRNA is upregulated, indicating differential regulation of the two separate VAMP genes and differential roles for the two VAMP isoforms in the regulation of exocytosis after nerve injury.

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.

Estrogen down-regulates mRNA encoding the exocytotic protein SNAP-25 in the rat pituitary gland.

Exocytosis is dependent on specific proteins that are located at the secretory granule membrane, in the cytoplasm or at the plasma membrane. The mRNA expression of synaptosomal-associated protein of 25 kDa (SNAP-25) isoforms SNAP-25a and SNAP-25b, vesicle associated membrane protein (VAMP) 2, mammalian homologue of unc-18 (munc-18) and Hrs-2 was studied in the pituitary of ovariectomized rats after subcutaneous insertion of capsules containing estrogen or placebo using in situ hybridization. Estrogen treatment (0.25 mg estradiol) significantly decreased SNAP-25a (32%; 10%) and SNAP-25b (25%; 22%) mRNA levels in the anterior and intermediate lobes, respectively, whereas VAMP-2, munc-18 and Hrs-2 mRNA levels remained unchanged. The results suggest that estrogen selectively regulates SNAP-25 transcription in the pituitary gland, but leaves VAMP-2, munc-18 and Hrs-2 mRNA levels unaffected.

Decreased mRNA levels for exocytotic proteins in the pituitary of aged rats.

Several protein components that are involved in the molecular regulation of transmitter release have been identified in neuronal, neuroendocrine and endocrine tissues. The expression of VAMP-2 (vesicle-associated membrane protein), munc-18 (mammalian homologue of unc-18) and SNAP-25 (synaptosomal-associated protein of 25 kDa) mRNA was studied in the rat anterior and intermediate pituitary gland of adult (2 months) and old (24 months) rats using in situ hybridization. In the pituitary anterior lobe of aged rats, there was a significant decrease in VAMP-2 (33%), munc-18 (17%) and SNAP-25 (20%) mRNA as compared to adult rats. In the intermediate lobe, there was a significant decrease in VAMP-2 (48%) and SNAP-25 (32%) mRNA of aged rats, whereas munc-18 mRNA levels were not significantly changed. Pituitaries from aged rats showed an increase in size which was paralleled by a significant decrease in the number of cells per unit area in the intermediate lobe, whereas the number was unaltered in the anterior lobe. The results suggest a genuine decrease in mRNA for exocytotic protein mRNA in the anterior pituitary, but that part of the decrease in the expression of VAMP-2 and SNAP-25 mRNA in the intermediate lobe can be explained by a decreased number of cells per unit area. The decline in anterior pituitary hormone secretion reported in aged rats appears to be parallelled by a down-regulation in mRNA levels for several proteins involved in the molecular regulation of exocytosis.

Botulinum neurotoxin F, a VAMP-specific endopeptidase, inhibits Ca(2+)-stimulated GH secretion from rat pituitary cells.

Botulinum neurotoxin F (BoNTx F) is a zinc-dependent endopeptidase that causes proteolytic cleavage of the vesicle protein VAMP (vesicle-associated membrane protein). VAMP is an important component of the molecular machinery regulating docking and fusion of secretory vesicles with the target membrane. We have investigated presence of VAMP protein in cultured rat anterior pituitary cells. Confocal laser microscopy revealed presence of VAMP-like immunoreactivity in secretory granules of GH-containing cultured rat anterior pituitary cells. Using BoNTx F, we have investigated whether VAMP is involved in growth hormone (GH) secretion. Treatment of streptolysin-O permeabilized GH-secreting cells with BoNTx F (2.0 and 20 nM) significantly inhibited Ca(2+)-induced GH release. The results show that the secretory granules of rat anterior pituitary cell contain VAMP protein and suggest that VAMP is of importance in regulating Ca(2+)-mediated GH secretion.

Effects of routinely given pethidine during labour on infants' developing breastfeeding behaviour. Effects of dose-delivery time interval and various concentrations of pethidine/norpethidine in cord plasma.

A standard dose of 100 mg of pethidine was given im to 13 healthy primiparae during labour. The aim of the study was to investigate whether developing breastfeeding behaviour in the newborn infant was associated with the dose-delivery time interval (DDI) or with the plasma concentration of pethidine and norpethidine in mixed cord blood at birth. The DDI was found to be unevenly distributed with no pethidine exposures in the time interval 5.4-8 h. The material was therefore divided into a "short DDI" group (1.1-5.3 h) and a "long DDI" group (8.1-9.9 h). The infants in the "short DDI" group had a depressed sucking behaviour in 15-45 min of observation and a delayed initiation of lip and mouth movements when compared with the infants in the "long DDI" group. Six of the thirteen infants did not suck their mothers' breasts during the observation period. These infants had higher median plasma concentrations of pethidine at birth than the seven infants who did start sucking. No differences wer found between the plasma levels of norpethidine and the behaviour. It was concluded that 100 mg of pethidine im as an analgesic given under routine conditions may have unfavourable effects on infants' developing breastfeeding behaviour if the DDI is short.

High uptake of [2-11C]acetyl-L-carnitine into the brain: a PET study.

The brain uptake of acetylcarnitine was investigated in rhesus monkeys using different position labeled acetyl-L-carnitine and related molecules with 11C by positron emission tomography. The uptake values of radio-labeled acetylcarnitine into the brain were quite different depending on the labeling positions of 11C. That is, the uptake values of L-[methyl-11C]carnitine and acetyl-L-[methyl-11C]carnitine were almost the same and extremely low, while the uptake of [1-11C]-acetyl-L-carnitine was slightly higher. The uptake value of [2-11C]acetyl-L-carnitine was by far the highest among the 11C-labeled acetyl-L-carnitine and L-carnitine. The uptake of [2-11C]acetyl-L-carnitine into the brain was suppressed by the intravenous administration of glucose. These results suggest that endogenous serum acetyl-L-carnitine has some roles on conveying an acetyl moiety into the brain especially under an energy crisis, and that an unknown metabolic pathway of [2-11C]acetyl moiety might be rather active in the brain.

Exocytotic proteins in enterochromaffin-like (ECL) cells of the rat stomach.

Proteins participating in vesicular docking and fusion have been identified in the nervous system. Such proteins appear to be important for the molecular regulation of exocytosis also in non-neuronal cells. The enterochromaffin-like (ECL) cells of the gastric acid-secreting (oxyntic) mucosa secrete histamine and chromogranin A-derived peptides, such as pancreastatin. Using immunohistochemistry, we have examined whether the ECL cells of the rat stomach, identified with antibodies to histidine decarboxylase (HDC, the histamine-forming enzyme), express the same exocytotic proteins as neurons. The ECL cells displayed immunoreactivity for synaptophysin, synaptotagmin III, vesicle-associated membrane protein-2 (VAMP-2), cysteine string protein (CSP), vesicular monoamine transporter-2 (VMAT-2), synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin, and Munc-18, but not for synaptotagmin I/II and VAMP-1. Synaptophysin and VMAT-2 could be detected not only in the ECL cells, but also in a population of HDC-negative cells. The demonstration of synaptotagmin III in only a limited number of ECL cells suggests the existence of a subpopulation of ECL cells. The results show that several exocytotic proteins, previously identified in neurons, are present in rat stomach ECL cells. Hence, proteins engaged in vesicular docking and in the fusion of granule/vesicle membrane with plasma membrane seem to exist in both neurons and endocrine cells.

Methodological aspects for in vitro characterization of receptor binding using 11C-labeled receptor ligands: a detailed study with the benzodiazepine receptor antagonist [11C]Ro 15-1788.

As a complement to in vivo studies with positron emission tomography (PET), it is desirable to perform in vitro characterization of newly developed 11C tracers. In this report we describe the technique for determination of receptor-ligand kinetics utilizing ligands labeled with the short-lived radionuclide 11C. The limitations and advantages are discussed. The benzodiazepine antagonist [11C]Ro 15-1788 was used as a model substance, and the use of storage phosphor plates for quantification of radioactivity was validated. Storage phosphor plates showed an excellent linear range (approximately 10[3]) and acceptable resolution (approximately 0.5 mm). Receptor-ligand kinetics, including depletion, association and dissociation, saturation and displacement were evaluated with good results through the use of short-lived radiotracers and storage phosphor plates.