The translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF), is encoded by a gene (Tpt1) that is highly conserved throughout phylogeny. TCTP is implicated in cell growth, acute allergic response, and apoptosis. In the present study, seven putative Tpt1 genes with different chromosomal localizations were identified in the mouse genome. In six of them, analysis of the 5' and 3' untranslated regions revealed the presence of flanking direct repeats and residual poly(A) tails typical of pseudogenes. Only three of the seven genes can produce a protein of the expected molecular weight. We isolated the genomic DNA of these three genes to analyze their sequence, genomic organization, and in vitro promoter activity. We found that mouse Tpt1 is localized on chromosome 14 with a canonical intron-exon organization, a functional promoter, and only one transcript that is ubiquitously expressed in all tissues.

Hereditary recurrent fevers (HRF) are a group of rare monogenic diseases leading to recurrent inflammatory flares. A large number of variants has been described for the four genes associated with the best known HRF, namely MEFV, NLRP3, MVK, TNFRSF1A. The Infevers database ( http://fmf.igh.cnrs.fr/ISSAID/infevers ) is a large international registry collecting variants reported in these genes. However, no genotype-phenotype associations are provided, but only the clinical phenotype of the first patient(s) described for each mutation. The aim of this study is to develop a registry of genotype-phenotype associations observed in patients with HRF, enrolled and validated in the Eurofever registry.

Genotype-phenotype associations observed in all the patients with HRF enrolled in the Eurofever registry were retrospectively analyzed. For autosomal dominant diseases (CAPS and TRAPS), all mutations were individually analyzed. For autosomal recessive diseases (FMF and MKD), homozygous and heterozygous combinations were described. Mean age of onset, disease course (recurrent or chronic), mean duration of fever episodes, clinical manifestations associated with fever episodes, atypical manifestations, complications and response to treatment were also studied. Data observed in 751 patients (346 FMF, 133 CAPS, 114 MKD, 158 TRAPS) included in the Eurofever registry and validated by experts were summarized in Tables. A total of 149 variants were described: 46 TNFRSF1A and 27 NLRP3 variants, as well as various combinations of 48 MVK and 28 MEFV variants were available.

We provide a potentially useful tool for physicians dealing with HRF, namely a registry of genotype-phenotype associations for patients enrolled in the Eurofever registry. This tool is complementary to the Infevers database and will be available at the Eurofever and Infevers websites.

Despite the common usage of a canonical, data-independent, hemodynamic response function (HRF), it is known that the shape of the HRF varies across brain regions and subjects. This suggests that a data-driven estimation of this function could lead to more statistical power when modeling BOLD fMRI data. However, unconstrained estimation of the HRF can yield highly unstable results when the number of free parameters is large. We develop a method for the joint estimation of activation and HRF by means of a rank constraint, forcing the estimated HRF to be equal across events or experimental conditions, yet permitting it to differ across voxels. Model estimation leads to an optimization problem that we propose to solve with an efficient quasi-Newton method, exploiting fast gradient computations. This model, called GLM with Rank-1 constraint (R1-GLM), can be extended to the setting of GLM with separate designs which has been shown to improve decoding accuracy in brain activity decoding experiments. We compare 10 different HRF modeling methods in terms of encoding and decoding scores on two different datasets. Our results show that the R1-GLM model outperforms competing methods in both encoding and decoding settings, positioning it as an attractive method both from the points of view of accuracy and computational efficiency.

The simultaneous recording of electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) can be used to localize interictal epileptiform discharges (IEDs). Previous studies have reported varying degrees of concordance of EEG-fMRI with electroclinical findings. The aim of the present study is to evaluate to what extent this variability is determined by the analytical strategy or by the properties of the EEG data. For that purpose, 42 IED sets obtained in 29 patients with epilepsy were reanalyzed using a finite impulse response approach, which estimates the hemodynamic response function (HRF) from the data and allows non-causal effects. Cardiac effects were treated as additional confounders in the model. This approach was compared to the classical approach assuming a fixed HRF for each voxel in the brain. The performance of each method was assessed by comparing the fMRI results to the EEG focus. The flexible model revealed more significantly activated voxels, which resulted in more activated brain regions concordant with the EEG focus (26 vs. 16). Correction for cardiac effects improved the results in 7 out of the 42 data sets. Furthermore, design theory for event-related experiments was applied in order to determine the influence of the number of IEDs and their temporal distribution on the success of an experiment. It appeared that this success is highly dependent upon the number of IEDs present during the recording and less on their temporal spacing. We conclude that the outcome of EEG-fMRI can be improved by using an optimized analytical strategy, but also depends on the number of IEDs occurring during the recording.

OBJECTIVE

It is not known on which time scales the nonlinear respirocardial interactions occur. This work's aim is to quantitatively assess functional respirocardial organization during quiet and active sleep of healthy full-term neonates by autonomic information flow (AIF) without limitation on specific time scales. Representing respirocardial interactions on a global time scale AIF carries information on a wider scope of interdependencies than known linear and nonlinear measures described. It assesses the complexity of heart rate fluctuations (HRF) and respiratory movements (RM) and their interaction comprising both linear and nonlinear properties. Thus, we hypothesized AIF to characterize novel aspects of sleep state-dependent respirocardial interaction.

METHODS

RM and ECG-derived HRF of six healthy full-term neonates were studied. We analyzed their power spectra, coherence, auto- and cross-correlation and complexity estimated on local ("next sample" prediction) and global time scales (an integral over AIF predicting for all time lags in HRF and RM).

RESULTS

We found the global AIF of HRF and RM to differ significantly between active and quiet sleep in all neonates, whereas on a local time scale this applied to the HRF AIF only. HRF complexity was larger in quiet than in active sleep. Respirocardial interaction was less complex in quiet versus active sleep in the high frequency band only.

CONCLUSIONS

Complex sleep state-related changes of respirocardial interdependencies cannot be identified completely on the local time scale. Considering the global time scale of respirocardial interactions allows a more complete physiological interpretation with regard to the underlying autonomic dynamics.

BACKGROUND

To identify alterations of retinal capillary blood flow in the papillomacular area in preclinical diabetic retinopathy using the Heidelberg scanning laser Doppler flowmeter.

METHODS

Ten eyes from ten patients with type 2 diabetes and no lesions visible on fundus photography (level 10 of Wisconsin grading) and ten eyes from ten healthy subjects of similar age range were examined with the HRF. Intravisit reproducibility of retinal capillary blood flow measurements was assessed in normal subjects and in type 2 diabetic patients, comparing different measurement areas and different analysis procedures: (a) 10x10 pixel box with original software, (b) 10x10 pixel box with SLDF software, and (c) whole-scan analysis with SLDF software (automatic full-field perfusion image analysis).

RESULTS

Intravisit reproducibility for the whole-scan analysis in the papillomacular area was 3.52%, 4.81% and 4.60% for volume (VOL), flow (FLW) and velocity (VEL) respectively. Using this method, mean and SD values for retinal capillary blood-flow are 13.25+/-2.87, 214.58+/-55.30 and 0.74+/-0.17, for VOL, FLW and VEL for healthy eyes, comparing with 19.85+/-6.22, 360.87+/-158.70 and 1.20+/-0.48 in eyes with preclinical diabetic retinopathy (P<0.010, P<0.019 and P<0.015 respectively).

CONCLUSIONS

The HRF shows acceptable reproducibility when using whole-scan analysis in the papillomacular area. Retinal capillary blood VOL, FLW and VEL were particularly increased in five of the ten diabetic eyes examined, with values over the mean + 2SD of the control population, suggesting that eyes showing increased retinal capillary blood flow may indicate risk of progression.

Accurate vessel detection in retinal images is an important and difficult task. Detection is made more challenging in pathological images with the presence of exudates and other abnormalities. In this paper, we present a new unsupervised vessel segmentation approach to address this problem. A novel inpainting filter, called neighborhood estimator before filling, is proposed to inpaint exudates in a way that nearby false positives are significantly reduced during vessel enhancement. Retinal vascular enhancement is achieved with a multiple-scale Hessian approach. Experimental results show that the proposed vessel segmentation method outperforms state-of-the-art algorithms reported in the recent literature, both visually and in terms of quantitative measurements, with overall mean accuracy of 95.62% on the STARE dataset and 95.81% on the HRF dataset.

Functional near-infrared spectroscopy (fNIRS) is an emerging non-invasive brain imaging technique and measures brain activities by means of near-infrared light of 650-950 nm wavelengths. The cortical hemodynamic response (HR) differs in attributes at different brain regions and on repetition of trials, even if the experimental paradigm is kept exactly the same. Therefore, an HR model that can estimate such variations in the response is the objective of this research. The canonical hemodynamic response function (cHRF) is modeled by two Gamma functions with six unknown parameters (four of them to model the shape and other two to scale and baseline respectively). The HRF model is supposed to be a linear combination of HRF, baseline, and physiological noises (amplitudes and frequencies of physiological noises are supposed to be unknown). An objective function is developed as a square of the residuals with constraints on 12 free parameters. The formulated problem is solved by using an iterative optimization algorithm to estimate the unknown parameters in the model. Inter-subject variations in HRF and physiological noises have been estimated for better cortical functional maps. The accuracy of the algorithm has been verified using 10 real and 15 simulated data sets. Ten healthy subjects participated in the experiment and their HRF for finger-tapping tasks have been estimated and analyzed. The statistical significance of the estimated activity strength parameters has been verified by employing statistical analysis (i.e., t-value>> t critical and p-value < 0.05).

DNA double-strand breaks (DSBs) can be repaired via two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). Our previous work showed that exposure to abiotic stresses resulted in an increase in point mutation frequency (PMF) and homologous recombination frequency (HRF), and these changes were heritable. We hypothesized that mutants impaired in DSB recognition and repair would also be deficient in somatic and transgenerational changes in PMF and HRF. To test this hypothesis, we analyzed the genome stability of the Arabidopsis thaliana mutants deficient in ATM (communication between DNA strand break recognition and the repair machinery), KU80 (deficient in NHEJ) and RAD51B (deficient in HR repair) genes. We found that all three mutants exhibited higher levels of DSBs. Plants impaired in ATM had a lower spontaneous PMF and HRF, whereas ku80 plants had higher frequencies. Plants impaired in RAD51B had a lower HRF. HRF in wild-type, atm and rad51b plants increased in response to several abiotic stressors, whereas it did not increase in ku80 plants. The progeny of stressed wild-type and ku80 plants exhibited an increase in HRF in response to all stresses, and the increase was higher in ku80 plants. The progeny of atm plants showed an increase in HRF only when the parental generation was exposed to cold or flood, whereas the progeny of rad51b plants completely lacked a transgenerational increase in HRF. Our experiments showed that mutants impaired in the recognition and repair of DSBs exhibited changes in the efficiency of DNA repair as reflected by changes in strand breaks, point mutation and HRF. They also showed that the HR RAD51B protein and the protein ATM that recognized damaged DNA might play an important role in transgenerational changes in HRF.

Background Inhaled nitric oxide (iNO) is being increasingly used in preterm infants < 34 weeks with hypoxemic respiratory failure (HRF) and/or pulmonary hypertension (PH). Objective To evaluate the risk factors, survival characteristics, and lung histopathology in preterm infants with PH/HRF. Methods Retrospective chart review was conducted to determine characteristics of 93 preterm infants treated with iNO in the first 28 days and compared with 930 matched controls. Factors associated with survival with preterm HRF and smooth muscle actin from nine autopsies were evaluated. Results Preterm neonates treated with iNO had a higher incidence of preterm prolonged rupture of membrane (pPROM ≥ 18 hours), oligohydramnios and delivered by C-section. In infants treated with iNO, antenatal steroids (odds ratio [OR],3.7; confidence interval [CI], 1.2-11.3; p = 0.02), pPROM (OR, 1.001; CI, 1.0-1.004; p = 0.3), and oxygenation response to iNO (OR, 3.7; CI, 1.08-13.1; p = 0.037) were associated with survival. Thirteen infants with all three characteristics had 100% (13/13) survival without severe intraventricular hemorrhage (IVH)/periventricular leukomalacia (PVL) compared with 48% survival (12/25, p = 0.004) and 16% severe IVH/PVL without any of these factors. Severity of HRF correlated with increased smooth muscle in pulmonary vasculature. Conclusion Preterm infants with HRF exposed to antenatal steroids and pPROM had improved oxygenation with iNO and survival without severe IVH/PVL. Precisely targeting this subset may be beneficial in future trials of iNO.

The translationally controlled tumor protein (TCTP) can be secreted independently of the endoplasmic reticulum/Golgi pathway and has extrinsic activities when it is characterized as the histamine releasing factor (HRF). Despite its important role in allergies and inflammation, little is known about how extracellular TCTP affects cancer progression. In this study, we found that TCTP was overexpressed in the interstitial tissue of colorectal cancer (CRC) and its expression correlated with poor survival, high pathological grades and metastatic TNM stage in CRC patients. TCTP expression was greater in metastatic liver tissue than in primary tumors and was increased in highly invasive CRC cells. We demonstrated that the expression of TCTP was regulated by HIF-1α and its release was increased in response to low serum and hypoxic stress. Recombinant human TCTP (rhTCTP) promoted the migration and invasiveness of CRC cells in vitro and contributed to distant liver metastasis in vivo. Furthermore, rhTCTP activated Cdc42, phosphorylated JNK (p-JNK), increasing the translocation of p-JNK from the cytoplasm to the nucleus, as well as the secretion of MMP9. In addition, the expression of TCTP positively correlated with that of Cdc42 and p-JNK in clinical CRC samples. The silencing of Cdc42, JNK and MMP9 significantly inhibited the Matrigel invasion of rhTCTP-enhanced CRC cells. Collectively, these results identify a new role for extracellular TCTP as a promoter of CRC progression and liver metastases via Cdc42/JNK/MMP9 activation.

Activation detection in functional Magnetic Resonance Imaging (fMRI) typically assumes the hemodynamic response to neuronal activity to be invariant across brain regions and subjects. Reports of substantial variability of the morphology of blood-oxygenation-level-dependent (BOLD) responses are accumulating, suggesting that the use of a single generic model of the expected response in general linear model (GLM) analyses does not provide optimal sensitivity due to model misspecification. Relaxing assumptions of the model can limit the impact of hemodynamic response function (HRF) variability, but at a cost on model parsimony. Alternatively, better specification of the model could be obtained from a priori knowledge of the HRF of a given subject, but the effectiveness of this approach has only been tested on simulation data. Using fast BOLD fMRI, we characterized the variability of hemodynamic responses to a simple event-related auditory-motor task, as well as its effect on activation detection with GLM analyses. We show the variability to be higher between subjects than between regions and variation in different regions to correlate from one subject to the other. Accounting for subject-related variability by deriving subject-specific models from responses to the task in some regions lead to more sensitive detection of responses in other regions. We applied the approach to epilepsy patients, where task-derived patient-specific models provided additional information compared to the use of a generic model for the detection of BOLD responses to epileptiform activity identified on scalp electro-encephalogram (EEG). This work highlights the importance of improving the accuracy of the model for detecting neuronal activation with fMRI, and the fact that it can be done at no cost to model parsimony through the acquisition of independent a priori information about the hemodynamic response.

The Translational Controlled Tumour Protein TCTP (gene symbol TPT1, also called P21, P23, Q23, fortilin or histamine-releasing factor, HRF) is a highly conserved protein present in essentially all eukaryotic organisms and involved in many fundamental cell biological and disease processes. It was first discovered about 35 years ago, and it took an extended period of time for its multiple functions to be revealed, and even today we do not yet fully understand all the details. Having witnessed most of this history, in this chapter, I give a brief overview and review the current knowledge on the structure, biological functions, disease involvements and cellular regulation of this protein.TCTP is able to interact with a large number of other proteins and is therefore involved in many core cell biological processes, predominantly in the response to cellular stresses, such as oxidative stress, heat shock, genotoxic stress, imbalance of ion metabolism as well as other conditions. Mechanistically, TCTP acts as an anti-apoptotic protein, and it is involved in DNA-damage repair and in cellular autophagy. Thus, broadly speaking, TCTP can be considered a cytoprotective protein. In addition, TCTP facilitates cell division through stabilising the mitotic spindle and cell growth through modulating growth signalling pathways and through its interaction with the proteosynthetic machinery of the cell. Due to its activities, both as an anti-apoptotic protein and in promoting cell growth and division, TCTP is also essential in the early development of both animals and plants.Apart from its involvement in various biological processes at the cellular level, TCTP can also act as an extracellular protein and as such has been involved in modulating whole-body defence processes, namely in the mammalian immune system. Extracellular TCTP, typically in its dimerised form, is able to induce the release of cytokines and other signalling molecules from various types of immune cells. There are also several examples, where TCTP was shown to be involved in antiviral/antibacterial defence in lower animals. In plants, the protein appears to have a protective effect against phytotoxic stresses, such as flooding, draught, too high or low temperature, salt stress or exposure to heavy metals. The finding for the latter stress condition is corroborated by earlier reports that TCTP levels are considerably up-regulated upon exposure of earthworms to high levels of heavy metals.Given the involvement of TCTP in many biological processes aimed at maintaining cellular or whole-body homeostasis, it is not surprising that dysregulation of TCTP levels may promote a range of disease processes, foremost cancer. Indeed a large body of evidence now supports a role of TCTP in at least the most predominant types of human cancers. Typically, this can be ascribed to both the anti-apoptotic activity of the protein and to its function in promoting cell growth and division. However, TCTP also appears to be involved in the later stages of cancer progression, such as invasion and metastasis. Hence, high TCTP levels in tumour tissues are often associated with a poor patient outcome. Due to its multiple roles in cancer progression, TCTP has been proposed as a potential target for the development of new anti-cancer strategies in recent pilot studies. Apart from its role in cancer, TCTP dysregulation has been reported to contribute to certain processes in the development of diabetes, as well as in diseases associated with the cardiovascular system.Since cellular TCTP levels are highly regulated, e.g. in response to cell stress or to growth signalling, and because deregulation of this protein contributes to many disease processes, a detailed understanding of regulatory processes that impinge on TCTP levels is required. The last section of this chapter summarises our current knowledge on the mechanisms that may be involved in the regulation of TCTP levels. Essentially, expression of the TPT1 gene is regulated at both the transcriptional and the translational level, the latter being particularly advantageous when a rapid adjustment of cellular TCTP levels is required, for example in cell stress responses. Other regulatory mechanisms, such as protein stability regulation, may also contribute to the regulation of overall TCTP levels.

Estimation and inferences for the hemodynamic response functions (HRF) using multi-subject fMRI data are considered. Within the context of the General Linear Model, two new nonparametric estimators for the HRF are proposed. The first is a kernel-smoothed estimator, which is used to construct hypothesis tests on the entire HRF curve, in contrast to only summaries of the curve as in most existing tests. To cope with the inherent large data variance, we introduce a second approach which imposes Tikhonov regularization on the kernel-smoothed estimator. An additional bias-correction step, which uses multi-subject averaged information, is introduced to further improve efficiency and reduce the bias in estimation for individual HRFs. By utilizing the common properties of brain activity shared across subjects, this is the main improvement over the standard methods where each subject's data is usually analyzed independently. A fast algorithm is also developed to select the optimal regularization and smoothing parameters. The proposed methods are compared with several existing regularization methods through simulations. The methods are illustrated by an application to the fMRI data collected under a psychology design employing the Monetary Incentive Delay (MID) task.

OBJECTIVE

To assess reliability and reproducibility of different analysis methods for retinal capillary flow, volume, and velocity from scanning laser Doppler flowmetry (SLDF) topography.

METHODS

SLDF topography analysis using the default retinal flowmeter (HRF) were compared to that using automatic full-field perfusion image analyzer (AFFPIA) and quantified as intraclass correlation coefficients (ICC).

RESULTS

The AFFPIA full-field method had the highest reliability, with ICC 0.99 for capillary flow. The reproducibility using the AFFPIA full-field method was high ICC 0.74 for capillary flow.

CONCLUSIONS

The AFFPIA full-field method is highly reliable and superior to the default HRF software.

OBJECTIVE

In vitro models suggest that Heidelberg retina flowmeter (HRF) measurements are affected by changes in photodetector sensitivity. We measured blood flow in a single volume of human retinal tissue in vivo at various sensitivity (DC) levels.

METHODS

The peripapillary retinal regions of 12 normal subjects were examined by HRF under five different sensitivity settings: (1) average DC range below 100; (2) average DC range below 125; (3) average DC range near 150 (normal sensitivity); (4) average DC range above 175; and (5) average DC range above 200 or extremely overexposed. The distributions of flow values were examined by pointwise analysis. All pixels from a common tissue location were analyzed, and the effect of their brightness on the flow measurement was evaluated by ANOVA with Fisher's protected least significant difference model.

RESULTS

ANOVA analysis of image DC level showed that significantly different DC levels were achieved for each of the five sensitivity settings (P < 0.0001). Flow values decreased with increasing DC for each of the 25th percentile, 50th percentile (P: < 0.0001 for each), 75th percentile (P: = 0.0026), 90th percentile (P: = 0.0216), and mean (P: = 0.0004) flow values. The percentage of pixels with values of zero (avascular tissue) increased with increasing photodetector sensitivity (P< 0.0001).

CONCLUSIONS

Improper sensitivity settings alter the detected percentage of avascular tissue and the blood flow measurements in tissue containing capillaries. Consistent assessment of retinal blood flow requires consistent photodetector sensitivity settings between longitudinal images.

This paper presents a novel, completely unsupervised fMRI brain mapping method that addresses the three problems of hemodynamic response function (HRF) variability, hemodynamic event timing, and fMRI response non-linearity. Spatial and temporal information are directly taken into account into the core of the activation detection process. In practice, activation detection at voxel v is formulated in terms of temporal alignment between sequences of hemodynamic response onsets (HROs) detected in the fMRI signal at v and in the spatial neighborhood of v, and the input sequence of stimuli or stimulus onsets. Event-related and epoch paradigms are considered. The multiple event sequence alignment problem is solved within the probabilistic framework of hidden Markov multiple event sequence models (HMMESMs), a new class of hidden Markov models. Results obtained on real and synthetic data significantly outperform those obtained with the popular statistical parametric mapping (SPM2) method without requiring any prior definition of the expected activation patterns, the HMMESM mapping approach being completely unsupervised.

Functional magnetic resonance imaging (fMRI) is a powerful and broadly used means of non-invasively mapping human brain activity. However fMRI is an indirect measure that rests upon a mapping from neuronal activity to the blood oxygen level dependent (BOLD) signal via hemodynamic effects. The quality of estimated neuronal activity hinges on the validity of the hemodynamic model employed. Recent work has demonstrated that the hemodynamic response has non-separable spatiotemporal dynamics, a key property that is not implemented in existing fMRI analysis frameworks. Here both simulated and empirical data are used to demonstrate that using a physiologically based model of the spatiotemporal hemodynamic response function (stHRF) results in a quantitative improvement of the estimated neuronal response relative to unphysical space-time separable forms. To achieve this, an integrated spatial and temporal deconvolution is established using a recently developed stHRF. Simulated data allows the variation of key parameters such as noise and the spatial complexity of the neuronal drive, while knowing the neuronal input. The results demonstrate that the use of a spatiotemporally integrated HRF can avoid "ghost" neuronal responses that can otherwise be falsely inferred. Applying the spatiotemporal deconvolution to high resolution fMRI data allows the recovery of neuronal responses that are consistent with independent electrophysiological measures.

A soluble form of homologous restriction factor (HRF) has been isolated from the cytoplasmic granules of human large granular lymphocytes that were cultured in the presence of recombinant interleukin 2 for 2-3 weeks. The granule-derived protein (approximately 65 kDa) is soluble in detergent-free solution and reacts with antibody produced to membrane HRF. HRF was first described as a 65-kDa membrane protein of human erythrocytes capable of inhibiting the formation of transmembrane channels by the membrane attack complex of complement. It has also been isolated from activated human lymphocytes and shown to confer upon these cells relative resistance to lysis by the membrane attack complex and by the complement component C9-related protein of human cytotoxic lymphocytes. The soluble HRF of lymphocyte granules inhibits reactive lysis of erythrocytes by the membrane attack complex of human complement. It was also found to be a potent inhibitor of (i) the cytolytic activity of the C9-related protein of human cytotoxic lymphocytes, (ii) human large granular lymphocyte cytotoxicity, and (iii) the cytotoxic activity of human CD8+ lymphocytes obtained by cell sorting from recombinant interleukin 2-activated peripheral blood mononuclear cells. It is proposed that granule-derived soluble HRF and cell surface-membrane-bound HRF are involved in the mechanism of self-protection of killer lymphocytes.

OBJECTIVE

To evaluate the intraobserver reproducibility of a software designed to assess retinal blood flow with the Heidelberg Retina Flowmeter (HRF).

METHODS

Ten subjects were consecutively recruited, and one eye of each patient was randomly selected for study. Blood flow measurements were analyzed by using an automatic full field perfusion image analysis (AFFPIA) program, which calculates the Doppler frequency shift and hemodynamic variables (flow, volume, and velocity) for each pixel. The resulting perfusion image is processed with respect to underexposed and overexposed pixels, saccades, and retinal vessel tree. Intraobserver reproducibility was calculated for the AFFPIA program. All the optic nerve heads were horizontally divided into three sections (superior, central, and inferior). The retinal blood flow was calculated in the superior and inferior section, and each section was further divided into three areas (temporal, nasal, and rim). The blood flow was evaluated for each area.

RESULTS

When the same observer analyzed the same image five times (intraobserver intraimage reproducibility), the AFFPIA coefficient of variation ranged from 0.5% to 5% in the temporal area, from 0.1% to 5.3% in the nasal area, and from 0.5 to 28% in the rim area. When the same observer analyzed three different images of the same section once (intraobserver interimage reproducibility), the AFFPIA coefficient of variation of flow measurements ranged from 1% to 7.3% in the temporal area, from 1.5% to 10% in the nasal area, and from 2 to 30% in the rim area.

CONCLUSIONS

Retinal blood flow measured by HRF and analyzed by AFFPIA had good intraobserver reproducibility. The reproducibility was significantly better in the temporal and nasal areas than in the rim area.

OBJECTIVE

The Heidelberg Retina Flowmeter (HRF; Heidelberg Engineering GmbH, Heidelberg, Germany) is a new instrument that determines hemodynamic variables at discrete locations of the retina and the optic disc. The objective of this study was to evaluate the influence of various HRF recording settings on the long-term variability of the HRF parameter. "Flow," computed at the optic nerve head in healthy individuals.

METHODS

The authors obtained 2 sets of 5 HRF recordings in 10 healthy individuals (age range, 23-60 years). The HRF recordings were obtained within a scan area of 10 degrees x 2.5 degrees (set 1) and 20 degrees x 5 degrees (set 2). For each set, the HRF recordings were obtained on 5 consecutive days. Respective HRF recordings for both sets were obtained on the same days. On these recordings, the HRF parameter, "Flow," was computed at 3 different regions of interest (temporal superior, temporal inferior, and temporal rim of the optic disc). At all three locations, Flow was computed within windows of measurement of 10 pixels x 10 pixels and 20 pixels x 20 pixels. The effect of larger windows (30 pixels x 30 pixels, 40 pixels x 40 pixels, and 50 pixels x 50 pixels) was tested at the temporal rim of the optic disc.

RESULTS

The highest reliability coefficient was reached with a scan area of 20 degrees x 5 degrees at the temporal superior rim of the optic disc (r = 0.93). Within a scan area of 20 degrees x 5 degrees, the size of the user-defined measuring window did not influence the reliability. Two models of analysis of variance disclosed that the only effect on the computed value of Flow that reached statistical significance was that because of the scan area (F = 11.172; p = 0.001). The location of the window of measurement and its size had no statistically significant effect.

CONCLUSIONS

The present results show that the location of the window of measurement has an important effect on the long-term variability of the HRF parameter, Flow. In addition, different scan areas influence significantly the computed values of this parameter.

Hereditary recurrent fevers (HRF) are rare diseases caused by molecular defects in genes involved in the regulation of innate immunity. Sixty-seven international laboratories participated in an external quality assessment (EQA) scheme, which was developed to appraise the accuracy of genetic testing. Reports were evaluated for the 12 items recommended by the OECD (Organisation for Economic Co-Operation and Development) guidelines for molecular diagnostics. The best documented items were the name of the gene, the biologist, or the patient, whereas information on the test and screening limits, and clinical interpretation of the disease inheritance were scarcely provided. The mutation nomenclature was incomplete in about 70% of the cases. In the first 2 years of EQA, we identified almost 30% genotyping error rate, which decreased markedly in the last year. The combined performance on the basis of the correct identification of all genotypes by a given laboratory in all the 3 years was only 40%, showing a critical need for improvement.

Histamine releasing factors (HRF) are a group of cytokines that release histamine and other mediators from mast cells and basophils. It has been speculated that HRF might play a major role in the pathogenesis of allergic diseases. Most investigators have studied PBMC as a source of HRF. This study was undertaken to investigate the cellular origin of HRF. Peripheral blood was processed to isolate and purify monocytes, T cells, CD4- T cells, CD8- T cells and B cells by using plastic adherence, 2-aminoethylisothiomonium-treated SRBC rosetting and negative selection with the use of mAb OKM1, OKT11, OKT8, OKT4, and OKB7 plus C. Highly purified subpopulations of PBMC were cultured alone or in the presence of Con A for 24 h. Supernatants were harvested, dialyzed, and assayed for HRF activity in the basophil histamine release test. We found that all subpopulations of PBMC including T cells, CD4- T cells, CD8- T cells, B cells, and monocytes produce variable quantities of HRF. The spontaneous production is very high in B cells but only barely measurable in T cells and monocytes. The synthesis of HRF by B cells was confirmed by abolishing the release of the activity after treatment of B cells with OKB7 mAb and C. Stimulation of cell populations by Con A significantly enhances HRF production by PBMC and T cells but not by B cells and monocytes. In mixing experiments, unstimulated monocytes + B cells showed synergism, but other combinations demonstrated an additive effect. This is the first demonstration of HRF production by human peripheral blood B cells. The results of this study also suggest that histamine releasing cytokines are of multiple cellular origin. This perhaps contributes to their molecular heterogeneity.

We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product.