Choice of immunoassay to evaluate porcine cytokine levels.

BACKGROUND: In order to adequately monitor cytokines in experimental models, currently available methods and commercially available kits should be compared. AIM: To compare the plasma and tissue concentrations of IL-1ß, IL-6, IL-8, IL-10, and TNF as a measure of systemic inflammation in septic pigs. METHODS: Cytokines were quantified from blood and tissue samples obtained at 0, 60, 120, 180, and 240 min, and in postmortem biopsies of the liver, kidney, lung, heart, and spleen from 26 anesthetized landrace pigs. (24 with experimental sepsis, two sham controls). Porcine-specific ELISAs (R&D) and multiplex (9-plex from Thermo Fischer, 13-plex from Millipore) immunoassays were compared. RESULTS: The assays differed for the different cytokines and between blood and tissue. In blood, the highest concentration of TNF and IL-6 was in ELISA, IL-1ß equal in ELISA and 13-plex, IL-8 in 13-plex and IL-10 in 9-plex. In tissue, the highest concentration of TNF and IL-1ß was in ELISA, IL-6 and IL-8 in 13-plex and IL-10 in 9-plex. CONCLUSION: The choice of analysis impacts the quantified cytokine responses in porcine models. ELISA and multiplex techniques supplement each other and our data suggest which assays to use for the quantification of the different cytokines.

On separating the magnocellular from the parvocellular: Responses to two statements by Goodhew et al.

Goodhew et al. (Attention Perception & Psychophysics, 79, 1147-1164, 2017) claim we (Skottun & Skoyles) hold: (1) that it is not possible to separate contributions from the magno- and parvocellular systems to psychophysical tasks, and (2) that there are no differences between magno- and parvocellular cells. Neither of these claims is correct.

Combined inhibition of C5 and CD14 efficiently attenuated the inflammatory response in a porcine model of meningococcal sepsis.

BACKGROUND: Fulminant meningococcal sepsis, characterized by overwhelming innate immune activation, mostly affects young people and causes high mortality. This study aimed to investigate the effect of targeting two key molecules of innate immunity, complement component C5, and co-receptor CD14 in the Toll-like receptor system, on the inflammatory response in meningococcal sepsis. METHODS: Meningococcal sepsis was simulated by continuous intravenous infusion of an escalating dose of heat-inactivated Neisseria meningitidis administered over 3 h. The piglets were randomized, blinded to the investigators, to a positive control group (n = 12) receiving saline and to an interventional group (n = 12) receiving a recombinant anti-CD14 monoclonal antibody together with the C5 inhibitor coversin. RESULTS: A substantial increase in plasma complement activation in the untreated group was completely abolished in the treatment group (p = 0.006). The following inflammatory mediators were substantially reduced in plasma in the treatment group: Interferon-γ by 75% (p = 0.0001), tumor necrosis factor by 50% (p = 0.01), Interleukin (IL)-8 by 50% (p = 0.03), IL-10 by 40% (p = 0.04), IL-12p40 by 50% (p = 0.03), and granulocyte CD11b (CR3) expression by 20% (p = 0.01). CONCLUSION: Inhibition of C5 and CD14 may be beneficial in attenuating the detrimental effects of complement activation and modulating the cytokine storm in patients with fulminant meningococcal sepsis.

A few words on differentiating magno- and parvocellular contributions to vision on the basis of temporal frequency.

A number of authors have proposed that changes in temporal frequency within the range of 0-30Hz may be used to differentiate contributions from the magno- and parvocellular systems. The present analyses estimate the percentage of active magnocellular cells as a function of frequency based on published cut-off values for magno- and parvocellular cells. These analyses indicate that varying the temporal frequency over the range of 0-30Hz has little effect upon the percentage of active magnocellular cells. The analyses were also carried out for a series of hypothetical cut-off frequencies and standard deviations of these frequencies for magnocellular cells. The results of these simulations indicate that even large alterations in these values do not alter the above conclusion to a noteworthy extent.

A few remarks on the utility of visual motion perception to assess the integrity of the magnocellular system or the dorsal stream.

It has been proposed that visual motion perception may be used to assess magnocellular or dorsal stream integrity. It is here pointed out, based on recently published data from dyslexic readers, that it is possible for deficient motion perception to exist without there being deficiencies in neither the magnocellular system nor in the dorsal stream. This makes it difficult to rely upon tests of motion perception to assess the integrity of these structures.

Massive Organ Inflammation in Experimental and in Clinical Meningococcal Septic Shock.

Fulminant meningococcal sepsis is characterized by a massive growth of bacteria in the circulation, regarded as the primary inflammatory site, with no specific solid organ focus. Here we aimed to study the local inflammatory response in organs using a porcine model of fulminant meningococcal septic shock challenged with exponentially increasing doses of heat inactivated Neisseria meningitidis. The results were compared with those obtained in organs post mortem from three patients with lethal meningococcal septic shock. Nine patients with lethal pneumococcal disease and 14 patients with sudden infant death syndrome served as controls. Frozen tissue were thawed, homogenized and prepared for quantification of bacterial DNA by real-time polymerase chain reaction, and key inflammatory mediators were measured by ELISA in the pig material and by multiplex in the human material. In addition, gene expression assayed by Affymetrix gene expression profiling was performed in the pig study. The porcine model revealed a major influx of N. meningitidis in lungs, liver, spleen, and kidneys accompanied with major production of cardinal inflammatory mediators including tumor necrosis factor, interleukin (IL)-1ß, IL-6, and IL-8, far exceeding the amount detected in blood. Genes encoding for these mediators revealed a similar profile. By comparing the wild-type with a lipopolysaccharide (LPS) deficient meningococcal strain, we documented that LPS was the dominant group of molecules inducing organ inflammation and was required for IL-8 production. IL-10 production was predominantly stimulated by non-LPS molecules. The massive organ inflammation in the porcine model was present in the three patients dying of meningococcal shock and differed markedly from the patients with lethal pneumococcal infections and sudden infant death syndrome. In conclusion, in meningococcal sepsis, a massive local inflammatory response occurs in specific organs.

On the use of spatial frequency to isolate contributions from the magnocellular and parvocellular systems and the dorsal and ventral cortical streams.

Many authors have claimed that suprathreshold achromatic stimuli of low and high spatial frequency can be used to separate responses from different entities in the visual system. Most prominently, it has been proposed that such stimuli can differentiate responses from the magnocellular and parvocellular systems. As is reviewed here, investigators who have examined stimulus specificity of neurons in these systems have found little difference between magno- and parvocellular cells. It has also been proposed that spatial frequency can be used to selectively activate the "magnocellular-dorsal stream". The present review indicates that cells in Area MT of the dorsal stream do prefer very low spatial frequencies. However, the review also shows that cells in Area V4 of the ventral stream respond, not only to relatively high spatial frequencies, but also to low frequency stimuli. Thus, low spatial frequencies cannot be relied upon to selectively activate the dorsal stream.

Metabolic Tumor Volume on 18F-FDG PET/CT Improves Preoperative Identification of High-Risk Endometrial Carcinoma Patients.

UNLABELLED: Our objective was to prospectively explore the diagnostic value of (18)F-FDG PET/CT for preoperative staging in endometrial carcinomas and to investigate whether (18)F-FDG PET-specific quantitative tumor parameters reflect clinical and histologic characteristics. METHODS: Preoperative (18)F-FDG PET/CT was prospectively performed on 129 consecutive endometrial carcinoma patients. Two physicians who did not know the clinical findings or staging results independently reviewed the images, assessing primary tumor, cervical stroma involvement and metastatic spread, and determining maximum and mean standardized uptake value (SUVmax and SUVmean, respectively) for tumor, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). All parameters were analyzed in relation to histomorphologic and clinical tumor characteristics. Receiver-operating-characteristic curves for identification of deep myometrial invasion and lymph node metastases were generated, and MTV cutoffs for predicting deep myometrial invasion and lymph node metastases were calculated. RESULTS: The sensitivity, specificity, and accuracy of (18)F-FDG PET/CT for the detection of lymph node metastases were 77%-85%, 91%-96%, and 89%-93%, respectively. SUVmax, SUVmean, MTV, and TLG were significantly related to deep myometrial invasion, presence of lymph node metastases, and high histologic grade (P < 0.015 for all) and independently predicted deep myometrial invasion (P < 0.015) and lymph node metastases (P < 0.025) after adjustment for preoperative histologic risk (based on subtype and grade) in endometrial biopsies. Optimal cutoffs for MTV in predicting deep myometrial invasion (20 mL) and the presence of lymph node metastases (30 mL) yielded odds ratios of 7.8 (P < 0.001) and 16.5 (P = 0.001), respectively. CONCLUSION: (18)F-FDG PET/CT represents a clinically valuable tool for preoperatively evaluating the presence of lymph node metastases in endometrial carcinoma patients. Applying MTV cutoffs for the prediction of deep myometrial invasion and lymph node metastases may increase diagnostic accuracy and aid preoperative identification of high-risk patients, enabling restriction of lymphadenectomy for patients with a low risk of aggressive disease.

The need to differentiate the magnocellular system from the dorsal stream in connection with dyslexia.

A number of authors have postulated a "magnocellular-dorsal stream" deficit in dyslexia. Combining the magnocellular system and the dorsal stream into a single entity in this context faces the problem that contrast sensitivity data do not point to a magnocellular deficiency linked to dyslexia, while, on the other hand, motion perception data are largely consistent with a dorsal stream dysfunction. Thus, there are data both for and against a "magnocellular-dorsal stream" deficit in connection with dyslexia. It is here pointed out that this inconsistency is abolished once it is recognized that the magnocellular system and the dorsal stream are separate entities.

A complement C5 gene mutation, c.754G>A:p.A252T, is common in the Western Cape, South Africa and found to be homozygous in seven percent of Black African meningococcal disease cases.

Patients with genetically determined deficiency of complement component 5 are usually diagnosed because of recurrent invasive Neisseria meningitidis infections. Approximately 40 individual cases have been diagnosed worldwide. Nevertheless, reports of the responsible genetic defects have been sporadic, and we know of no previous reports of C5 deficiency being associated with a number of independent meningococcal disease cases in particular communities. Here we describe C5 deficiency in seven unrelated Western Cape, South African families. Three different C5 mutations c.55C>T:p.Q19X, c.754G>A:p.A252T and c.4426C>T:p.R1476X were diagnosed in index cases from two families who had both presented with recurrent meningococcal disease. p.Q19X and p.R1476X have already been described in North American Black families and more recently p.Q19X in a Saudi family. However, p.A252T was only reported in SNP databases and was not associated with disease until the present study was undertaken in the Western Cape, South Africa. We tested for p.A252T in 140 patients presenting with meningococcal disease in the Cape Town area, and found seven individuals in five families who were homozygous for the mutation p.A252T. Very low serum C5 protein levels (0.1-4%) and correspondingly low in vitro functional activity were found in all homozygous individuals. Allele frequencies of p.A252T in the Black African and Cape Coloured communities were 3% and 0.66% and estimated homozygosities are 1/1100 and 1/22,500 respectively. In 2012 we reported association between p.A252T and meningococcal disease. Molecular modelling of p.A252T has indicated an area of molecular stress in the C5 molecule which may provide a mechanism for the very low level in the circulation. This report includes seven affected families indicating that C5D is not rare in South Africa.

A few observations on linking VEP responses to the magno- and parvocellular systems by way of contrast-response functions.

It has been proposed that magno- and parvocellular contributions to Visually Evoked Potentials (VEPs) can be isolated, or differentiated, by noting the contrast-response relationships of the responses. This suggestion is examined quantitatively by determining the similarity between various sets of VEP data that have been attributed to the magno- and parvocellular systems and previously reported contrast-response functions for different kinds of neurons (magno- and parvocellular neurons and V1, V4, and MT cells) and combinations of the contrast-response functions for these neurons. It is found that other neurons, or combinations of other neurons, typically give better fits to the data than do magno- and parvocellular cells. Thus, to attribute VEP responses to the magno- or parvocellular systems based on contrast-responses properties faces difficulties.

Subjective criteria and illusions in visual testing: some methodological limitations.

It is argued that illusions cannot generally be investigated with criterion-independent methods. This limits the value of the data obtained from them. This is particularly important when the results are compared between groups of subjects, for example, between dyslexic readers and controls, since it is possible that the differences between the groups reflect differences with regard to criteria rather than real perceptual differences.

Combined inhibition of complement C5 and CD14 markedly attenuates inflammation, thrombogenicity, and hemodynamic changes in porcine sepsis.

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli-induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin-antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1ß, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

On using very high temporal frequencies to isolate magnocellular contributions to psychophysical tasks.

In connection with dyslexia several authors have sought to employ stimuli of very high temporal frequency to isolate magnocellular contributions to visual tasks. It is here pointed out that considerable evidence indicate that the ability to see the very highest temporal frequencies is limited by cortical mechanisms. This suggests that variations and abnormalities in this ability may reflect cortical factors rather than magnocellular ones. It is therefore difficult to rely upon very high temporal frequency stimuli to isolate contributions from the magnocellular system.

Is there a role for PET-CT and SPECT-CT in pediatric oncology?

During the last decade, hybrid imaging has revolutionized nuclear medicine. Multimodal camera systems, integrating positron emission tomography (PET) or single photon emission computed tomography (SPECT) with computed tomography (CT) now combine the contrast provided by tumor-avid radioactive drugs with the anatomic precision of CT. While PET-CT to a great extent has replaced single-modality PET in adult oncology, the use of PET-CT in children has been controversial, since even the lowest dose CT protocols adds approximately 2 mSv to the radiation dose of about 4 mSv from the PET-study with F-18-fluorodeoxyglucose (F-18-FDG). The article describes the current techniques used, discusses radiation doses and gives an overview of current indications for PET-CT and SPECT-CT in children. Hybrid imaging with a tumor-avid radioactive drug provides extremely high contrast between tumor and background tissues, while the CT component helps to locate the lesion anatomically. Currently both PET-CT and SPECT-CT play a role in pediatric oncology; PET-CT using F-18-FDG particularly for staging and follow-up of lymphoma and brain cancer, bone and soft tissue sarcomas; SPECT-CT with I-123-metaiodobenzylguanidine (MIBG) for tumors of the sympathetic nervous system such as neuroblastoma and pheochromocytoma while the remaining neuroendocrine tumors are imaged with radioactively labeled somatostatin analogues. To reduce radiation dose, a low-dose CT in combination with ultrasound and/or magnetic resonance imaging for the assessment of anatomy is often preferred.