Blood virosphere in febrile Tanzanian children.

Viral infections are the leading cause of childhood acute febrile illnesses motivating consultation in sub-Saharan Africa. The majority of causal viruses are never identified in low-resource clinical settings as such testing is either not part of routine screening or available diagnostic tools have limited ability to detect new/unexpected viral variants. An in-depth exploration of the blood virome is therefore necessary to clarify the potential viral origin of fever in children. Metagenomic next-generation sequencing is a powerful tool for such broad investigations, allowing the detection of RNA and DNA viral genomes. Here, we describe the blood virome of 816 febrile children (<5 years) presenting at outpatient departments in Dar es Salaam over one-year. We show that half of the patients (394/816) had at least one detected virus recognized as causes of human infection/disease (13.8% enteroviruses (enterovirus A, B, C, and rhinovirus A and C), 12% rotaviruses, 11% human herpesvirus type 6). Additionally, we report the detection of a large number of viruses (related to arthropod, vertebrate or mammalian viral species) not yet known to cause human infection/disease, highlighting those who should be on the radar, deserve specific attention in the febrile paediatric population and, more broadly, for surveillance of emerging pathogens.Trial registration: ClinicalTrials.gov identifier: NCT02225769.

The minipterional approach for ruptured and unruptured anterior circulation aneurysms: Our initial experience.

OBJECTIVE: To report our experience with the minipterional (MPT) craniotomy approach for anterior circulation aneurysms and to discuss the clinical outcomes as well as to evaluate the advantages of this unique approach. MATERIALS AND METHODS: Single-center retrospective review of 57 cases involving anterior circulation aneurysms both ruptured and unruptured aneurysms treated with the MPT. We analyzed the clinical and patient demographic data, aneurysm characteristics, surgical outcomes, and complications in these individuals. RESULTS: Between July 2008 and March 2014, of the 57 patients reviewed: 45 had middle cerebral artery (MCA), 6 had internal carotid artery terminus, and 7 had posterior communicating artery aneurysms. 20 of the 57 patients presented with a ruptured aneurysm. The average aneurysm size was 5.8 mm. The length of hospitalization for unruptured aneurysm cases ranged between 3 and 5 days. The average follow-up for all cases was 21.5 months. Successful clipping of the aneurysms was obtained in all patients. None of the cases required additional skin incisions or craniotomy extensions. The overall surgical outcomes were favorable. There was no postoperative facial nerve damage, temporalis muscle wasting, or symptoms of paresthesias around the incision line. Two patients developed a postoperative stroke manifested as symptoms of unilateral arm and facial weakness, receptive aphasia, and dysarthria. CONCLUSION: The MPT provides a reliable and less invasive alternative to the standard pterional craniotomy. Furthermore, ruptured and unruptured anterior circulation aneurysms can safely and effectively be treated with limited bone removal which provides better cosmetic outcomes and excellent postoperative temporalis muscle function.

Role of squamosal suture as a consistent landmark for middle fossa approach craniotomy: an anatomical study.

Objective To establish a consistent surface bony landmark for a middle fossa approach (MFA) lateral craniotomy represented by the squamosal suture (SS). Methods In 60 dried skulls, we assessed the relation between the SS and the external auditory canal (EAC). The lateral portion of the middle cranial fossa floor was also assessed for a possible relation with the anteroposterior diameter (APD) of the squama temporalis (ST). Clinically, we applied our findings on the SS in MFA for different lesions. Results A vertical line at the EAC divided the ST into the anterior part constituting 61% of the APD (i.e., two thirds) and the posterior part forming 39% (i.e., one third). The average ST height was 35.92 mm. The SS posterior limit at the supramastoid crest was located just anterior to the external projection of the petrous ridge in 35 skulls (58%) and exactly corresponded to it in 25 skulls (42%). The APD of the ST equals on average 97% of the APD of the lateral middle cranial fossa. Optimum exposure of the middle fossa was obtained without any further craniotomy extension. Conclusion The SS serves as a consistent natural surface bony landmark for MFA. Optimum craniotomy, two thirds anterior to the EAC and one third posterior, is obtained following SS as a landmark.

S-nitroso albumin enhances bone formation in a rabbit calvaria model.

Nitric oxide (NO) is a mediator involved in bone regeneration. We therefore examined the effect of the novel NO donor, S-nitroso human serum albumin (S-NO-HSA) on bone formation in a rabbit calvaria augmentation model. Circular grooves (8 mm diameter, two per animal) were created by a trephine drill in the cortical bone of 40 rabbits and titanium caps were placed on the rabbit calvaria bone filled with a collagen sponge soaked with either 100 µL S-NO-HSA (5%, 20%) or human albumin (5%, 20%). After 4 weeks the titanium hemispheres were subjected to histological and histomorphometric analysis. Bone formation and the volume of the residual collagen sponge were evaluated. S-NO-HSA treatment groups had a significantly higher volume of newly formed bone underneath the titanium hemispheres compared to the albumin control groups (5%: 15.5 ± 4.0% versus 10.6 ± 2.9%; P < 0.05; 20%: 14.0 ± 4.6% versus 6.0 ± 3.8%; P < 0.01). The volume of residual collagen sponge was also significantly lower in the S-NO-HSA groups compared to the control groups (5%: 0.4 ± 0.5% versus 2.6 ± 2.4%; P < 0.05 and 20%: 1.5 ± 2.7% versus 13.0 ± 18.7%; P < 0.01). This study demonstrates for the first time that S-NO-HSA promotes bone formation by slow NO release. Additionally, S-NO-HSA increases collagen sponge degradation.

S-nitroso human serum albumin given after LPS challenge reduces acute lung injury and prolongs survival in a rat model of endotoxemia.

Endotoxemia leads to the induction of inducible nitric oxide synthase (NOS-2) and increased expression of numerous inflammatory mediators contributing to endotoxin-induced acute lung injury. We tested the hypothesis that supplementation of nitric oxide (NO) by the novel NO donor S-nitroso human serum albumin (S-NO-HSA) given after lipopolysaccharide (LPS) challenge may reduce NOS-2 expression, lung inflammation and acute lung injury. Rats were divided into four groups: sham-operated (no treatment), LPS, LPS+HSA (human serum albumin), and LPS+S-NO-HSA. LPS was administered intravenously (20 mg kg(-1)) resulting in acute lung injury and a high mortality rate within 6 h (>90%). LPS-induced lung injury was characterized by an increased lung edema (lung wet/dry weight ratio), pulmonary neutrophil infiltration (myeloperoxidase activity, MPO) as well as a robust inflammatory response [increased expression of intercellular adhesion molecule-1 (ICAM-1), NOS-2, and cyclooxygenase-2 (COX-2)]. Infusion of S-NO-HSA or HSA was started 2 h after LPS and continued for 4 h (total dose of 72 mg kg(-1)) at a rate of 300 microg kg(-1) min(-1). S-NO-HSA but not HSA prolonged survival of endotoxemic rats, reduced the hypotensive response to LPS, minimized LPS-induced lung edema and injury, normalized MPO activity as well as diminished lung expression of pro-inflammatory molecules such as ICAM-1, NOS-2, and COX-2. Continuous supplementation of NO by S-NO-HSA after LPS challenge prevents induction of NOS-2, provides significant protection of endotoxin-induced acute lung injury, and prevents early mortality in endotoxic shock in rats. Our results suggest a potential therapeutic role for S-NO-HSA in endotoxemia.

Ten polymorphic microsatellite loci for Allobates femoralis, an Amazonian dendrobatoid frog.

Within the anuran amphibians, dendrobatoids (poison-arrow frogs) are renowned for their parental care, but the lack of highly variable DNA markers so far precluded precise and comprehensive measurements of their genetic mating system. Here we present 10 polymorphic microsatellites for Allobates femoralis, a widespread dendrobatoid from the Amazon basin. In 24 field-collected individuals, we found between seven and 15 alleles per locus, without deviations from Hardy-Weinberg equilibrium or linkage disequilibrium. The loci will be employed to quantify reproductive success in view of male territoriality and female mate choice, as well as to determine the fine-scale genetic structure of local populations.

Continuous venovenous hemofiltration improves arterial oxygenation in endotoxin-induced lung injury in pigs.

BACKGROUND: Hypoxemia is common in septic acute lung failure. Therapy is mainly supportive, and most trials using specific inhibitors of key inflammatory mediators (ie., tumor necrosis factor alpha, interleukin 1) have failed to prove beneficial. The authors investigated if a nonspecific blood purification technique, using zero-balanced high-volume continuous venovenous hemofiltration (CWH), might improve arterial oxygenation in a fluid-resuscitated porcine model of endotoxin-induced acute lung injury. METHODS: Piglets of both sexes weighing 25-30 kg were anesthetized and mechanically ventilated. After baseline measurements, animals received an intravenous infusion of 0.5 mg/kg endotoxin (Escherichia coli lipopolysaccharide). One hour after endotoxin, animals were randomly assigned to either treatment with CWH (endotoxin + hemofiltration, n = 6) or spontaneous course (endotoxin, n = 6). At 4 h after randomization, animals were killed. Hemofiltration was performed from femoral vein to femoral vein using a standard circuit with an EF60 polysulphone hemofilter. RESULTS: Endotoxin challenge induced arterial hypoxemia, an increase in peak inspiratory pressure, pulmonary hypertension, and systemic hypotension. Treatment with CWH did not improve systemic or pulmonary hemodynamics. However, arterial oxygenation was increased in endotoxin-challenged animals at 5 h after completion of endotoxin infusion, as compared with animals not receiving CVVH (arterialoxygen tension, 268+/-33 vs. 176+/-67 mm/Hg, respectively, P < 0.01). In addition, treatment with CWH attenuated the endotoxin-induced increase in peak inspiratory pressure and increased lung compliance. CONCLUSION: These results suggest that nonspecific blood purification with high-volume CWH improves arterial oxygenation and lung function in endotoxin-induced acute lung injury in pigs, independent of improved hemodynamics, fluid removal, or body temperature.

Protection against endotoxemia in rats by a novel tetrahydrobiopterin analogue.

We studied the effects of a novel pterin antagonist of NO synthase, the 4-amino analogue of tetrahydrobiopterin (4-ABH4), in a rat model of endotoxic shock and compared its properties with those of N(G)-monomethyl L-arginine (L-NMMA). Treatment with a bolus dose of 4-ABH4 at 2 h after LPS challenge significantly improved the 6-day survival rate, compared with the controls treated with saline. L-NMMA treatment did not significantly influence the survival rate. This bolus treatment, using either compound, had no effect on the plasma nitrite + nitrate or plasma IL-6 levels. The continuous infusion of 4-ABH4 efficiently suppressed the enhanced calcium-dependent/independent NO synthase activities induced by endotoxin in lung homogenates and completely suppressed the increase in plasma nitrite + nitrate caused by endotoxin at 5 h, with no significant difference compared with the L- NMMA treatment. Treatment of RAW264.7 murine macrophages with 4-ABH4 but not with L-NMMA suppressed endotoxin-induced tumor necrosis factor-alpha release by the cells, whereas nitrite in the supernatant decreased in a dose-dependent fashion in both assay systems. Our data show that 4-ABH4, an inhibitor of inducible NO synthase, significantly improves survival in a rat model of endotoxic shock when administered in a bolus dose that does not reduce plasma total nitrite + nitrate levels. Because we observed no overt signs of toxicity and no influence on organ-specific tetrahydrobiopterin levels, we conclude that the novel compound 4-ABH4 is a promising drug candidate for protection against endotoxin-related mortality.

Oxidatively modified plasma phospholipids containing reactive carbonyl functions measured by HPLC: evidence for phosphatidylcholine-bound aldehydes in plasma of burn patients.

A HPLC method has been developed to measure phosphatidylcholine (PC) containing reactive carbonyl functions in the sn-acyl residue in order to study processes in which such reactive carbonyls can be formed due to e.g. oxidative fragmentation. The method has been applied to determine PC-bound carbonyls as 2,4-dinitrophenylhydrazones (DNPH) in plasma of burn patients. Plasma from healthy volunteers served as controls. Additionally, in vitro oxidation experiments (A: plasma, buffer diluted; B: plasma + iron-EDTA complex and C: plasma + iron-EDTA complex + H2O2) have been performed to obtain and to identify 2,4-dinitrophenylhydrazine derivatizable carbonyl functions in plasma PC. Both, the PC-aldehydes and PC-aldehyde DNPH derivatives were cleavable with phospholipase C. Quantification was based on thin-layer chromatography purified soybean phosphatidylcholine, which was identically oxidized and derivatized as the plasma lipids in vitro.

Exposure of the hydrophobic components of porcine lung surfactant to oxidant stress alters surface tension properties.

We have tested the hypothesis that oxidation of lung surfactant results in loss of surface tension lowering function. Porcine lung surfactant was exposed to conditions known to cause lipid peroxidation (0.2 mM FeCl2 + 0.1 mM H2O2 or 5 microM CuCl2). Lipid peroxidation was verified by detection of conjugated dienes, thiobarbituric acid reactive substances, fluorescent products, hydroxy alkenals, and loss of unsaturated fatty acids. Exposed samples had significantly diminished surface tension lowering ability in vitro as measured in a bubble surfactometer. Samples exposed to FeCl2 + H2O2 had significantly diminished surface tension lowering ability in vivo as indicated by their reduced ability to improve lung compliance of surfactant-deficient fetal rabbits. Oxidation of phospholipid mixtures with surface tension lowering activity and containing unsaturated acyl groups resulted in partial loss of activity as determined in vitro. These results suggest that the effect of oxidants on lung surfactant function is due, in part, to effects on the phospholipid components and that acute pulmonary inflammation accompanied by oxygen radical production may result in surfactant lipid peroxidation and loss of surface tension lowering function.

Free radical activity and loss of plasma antioxidants, vitamin E, and sulfhydryl groups in patients with burns: the 1993 Moyer Award.

This study examines the relationship of burn injury and plasma levels of conjugated dienes, total sulfhydryl groups, and vitamin E in patients with thermal injuries. Plasma neopterin levels were determined as an index of macrophage activity and serine elastase as an index of polymorphonuclear cell activation. Thirteen patients with burns, six survivors and seven nonsurvivors, were studied for the first 4 days, then every other day until postburn day 14. Twelve healthy volunteers served as the control group. Survivors had 56% +/- 4% total body surface area burned, and nonsurvivors had 63.9% +/- % total body surface area burned. The patients with burns, compared with the control group, showed elevated plasma levels of the lipid peroxidation products conjugated dienes (0.767 +/- 0.045 vs 0.269 +/- 0.013 Abs at 233 nm) and reduced levels of the natural scavengers of free radicals, vitamin E (196.2 +/- 12.6 vs 841.1 +/- 22.7 micrograms/dl) and total sulfhydryl groups (54.0 +/- 0.4 vs 15.8 +/- 1.0 mumol/dl). The total sulfhydryl groups/conjugated dienes ratio fell at a greater rate (9.8% +/- 3.2% vs 3.2% +/- 0.7%/day) in nonsurvivors than in survivors (p < 0.05 by Mann-Whitney). The levels of elastase were slightly elevated in the patients with burns, but there was no difference between survivors and nonsurvivors. Normal neopterin levels are 3 to 10 nm/L; peak levels were 119 +/- 48 nm/L in nonsurvivors and 37.4 +/- 10 nm/L in survivors. Patients with burns demonstrated evidence of increased oxygen free radical activity and activation of polymorphonuclear cell and macrophages. Nonsurvivors demonstrated increased consumption of antioxidants compared with survivors.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of oxygen radicals in shock related cell injury.

Shock-related organ failure evolves from a variety of starting points--ischemia, reperfusion, non-bacterial or bacterial inflammation--several mechanisms are involved. In addition to the effects of xanthine oxidase after ischemia/reperfusion, toxic oxygen species from phagocytes that accumulate in both intra- and extravascular tissue spaces are of central importance. A critical event is the contact (adhesion) of leukocytes to endothelial cells, which consequently are the targets for leukocyte products. Damage of membranes by lipid peroxidation and by exposure to mediators such as platelet activating factor (PAF), leukotrienes and proteases, leads to increased permeability, tissue oedema and organ dysfunction. Thus antioxidants and other agents that control phagocyte function are likely to contribute to the protection of the permeability barrier in shock states.

Characterization of phosphatidylcholines in rabbit lung lavage fluid by positive and negative ion fast-atom bombardment mass spectrometry.

The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M+H-2H]+ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.

Methodological approach to the characterization of diacylphosphatidyl cholines in rabbit lung lavage fluid by fast-atom bombardment mass spectrometry.

Strong evidence exists that the distribution of phospholipids in the lung is a function of the degree of adult respiratory distress syndrome. The capabilities of positive-ion fast-atom bombardment (FAB) mass spectrometry for the determination of the relative distribution of intact diacylphosphatidylcholine species in lung lavage fluid were investigated. Two different FAB matrices and two different isolation/purification procedures--extraction/thin layer chromatography--high performance liquid chromatography (TLC-HPLC)--have been evaluated. In addition the relative fatty acid composition of the diacylphosphatidylcholines was determined by negative-ion FAB mass spectrometry using the [RCOO]- fragment ion. These results were compared with those obtained by gas chromatographic determination of the fatty acid methylesters.

Normal phosphatidylcholine composition of lung surfactant 24 hours after inhalation injury.

The effects of cotton smoke injury on the phosphatidylcholine composition of pulmonary surfactant material were studied. While under halothane anesthesia, 18 ewes were intubated using a double-lumen tube. In six sheep the left lung was exposed to smoke; in another six sheep the right lung was exposed to smoke. A sham group of six sheep was insufflated with air instead of smoke. Twenty-four hours later the animals were killed, and alveolar lavage fluid was obtained from both lungs. Phospholipid material was separated, and its relative phosphatidylcholine composition was determined. In the smoke-treated lungs this composition was not different statistically when compared with the contralateral and the sham-exposed lungs. These findings suggested that smoke inhalation has no effect on the composition of the phosphatidylcholine fraction in the alveolar lining layer 24 hours after injury.

A preparative high performance liquid chromatography method for separation of lecithin: comparison to thin-layer chromatography.

We have developed a high performance liquid chromatography (HPLC) method to separate lecithin from other phospholipid classes and to obtain lecithin from biologic materials. The separation was performed on a preparative 10-micron Spherisorb column with an optimized solvent system consisting of the following components: acetonitrile, isopropanol, methanol, water, and trifluoroacetic acid. The advantages of this method are the use of an isocratic solvent system limited to about 30 min and the very good separation of the phosphatidyl-choline fraction from the sphingomyelin fraction. Furthermore, the HPLC method has a better recovery rate than the thin-layer chromatography method, and it can be run under automatic control.

Influence of parenteral nutrition on phospholipid metabolism in posttraumatic rat lungs.

In the current investigation, we studied two groups of rats--one group supplied exogenous phospholipid precursors (carbohydrate plus fat emulsion group) and the other given only calories (carbohydrate group)--to evaluate the effects on surfactant composition by normocaloric alimentation, using a hypovolemic-traumatic shock model. The total phospholipid (PHL) contents of lung tissue were similar in both groups. However, we found differences in the dipalmitoylphosphatidylcholine fraction (DPPC--the most important component of surfactant material) in both lung tissue and lavage fluid. With lipid emulsion, there was an increased fraction of saturated lecithins (mainly DPPC) both in lung tissue and lavage fluid, similar to former studies with hypocaloric alimentation. In this model, those findings suggest that the PHL pattern does not depend on the quantity of caloric supply, but, rather, on the infusion composition. The enhanced DPPC content is further reflected in improved surfactant status: lipid in parenteral nutrition (PN) may exert a direct salutary effect on lung mechanics.

[Influence of parenteral nutrition on phospholipid metabolism in the rat lung in the posttraumatic period]. / Beeinflussung des Phospholipidstoffwechsels der Rattenlunge in der posttraumatischen Phase durch parenterale Ernährung.

Most experimental studies in the field of parenteral nutrition following trauma are aimed at a reduction of catabolism and therefore an improvement of the N-balance. The aim of this study was to investigate the influence of PN on PHL composition in the posttraumatic lung, especially in the surfactant fraction. We compared a carbohydrate - amino acid formula with a formula containing carbohydrate - amino acid - lipid. Polytrauma was performed by single femur fracture plus laparotomy with eventeration of the intestines for 30 minutes and hypovolemia (2 ml/100 g bw). After an adaption phase with reduced caloric supply, full caloric support was given from the 4th to the 14th day (100 Kcal/kg bw/day). The following metabolic parameters were used: N-balance; phospholipid total, -classes, -molecular species and fatty acid pattern of lecithin in lung tissue and lavage (using special HPLC and GC techniques). There were small differences between the groups using standard metabolic parameters (such as nitrogen balance). In contrast, we found significantly altered phospholipid patterns in the lung. With lipid emulsion there was an increased fraction of saturated lecithins (mainly dipalmitoyl phosphatidylcholine). From our data we conclude that it is possible to influence surfactant phospholipid composition by parenteral nutrition through the use of lipid emulsion. This might be useful for posttraumatic lung treatment.