Leukocytoclastic vasculitis in children: clinical characteristics, subtypes, causes and direct immunofluorescence findings of 56 biopsy-confirmed cases.

BACKGROUND: Leukocytoclastic vasculitis (LCV) in children is a complex group of conditions. OBJECTIVES: This study presents the demographics, clinical features, direct immunofluorescence (DIF) results and suspected aetiologies of 56 biopsy-confirmed cases of leukocytoclastic vasculitis in children. METHODS: Retrospective review of 56 children seen at Mayo Clinic in Rochester, Minnesota, from 1993 to 2013 with clinical features and cutaneous biopsy consistent with LCV. RESULTS: Twenty-seven (48%) cases were found to be due to IgA vasculitis (Henoch-Schonlein purpura). The remaining cases were found to be due to cutaneous small-vessel vasculitis (n = 19, 34%), urticarial vasculitis (n = 5, 9%), ANCA-associated vasculitis (n = 4, 7%) and acute haemorrhagic oedema of infancy (n = 1, 2%). IgA vasculitis was found to be associated with abdominal pain (P = 0.008), whereas the non-IgA vasculitis group was associated with headache (P = 0.052). Children with IgA vasculitis had palpable purpura (P = <0.001), petechia (P = 0.057), vesicles (P = 0.009) and involvement of the buttock (P = 0.004) more frequently than the non-IgA vasculitis group. On DIF, perivascular IgA was positive in IgA vasculitis compared to non-IgA vasculitis cases (P = <0.001), the other conjugates were similar between the two groups. CONCLUSION: The most common subtype of biopsy-confirmed LCV in children is IgA vasculitis. Clinical features, exam characteristics and DIF results can be helpful in determining the subtype of cutaneous vasculitis in children.

Intercellular transfer and supramolecular organization of human leukocyte antigen C at inhibitory natural killer cell immune synapses.

After accumulation of target cell human leukocyte antigen (HLA)-C at inhibitory natural killer (NK) cell immune synapses, some HLA-C transfers from target cells to NK cell plasma membranes and cytoplasm. This unexpected intercellular transfer of HLA-C is dependent on NK receptor recognition, since HLA-Cw6 or -Cw4 but not -Cw3 transfer to an NK transfectant expressing killer Ig-like receptor (KIR)2DL1. Strikingly, live-cell time-lapse laser scanning confocal microscopy shows vesicles containing target cell green fluorescent protein-tagged HLA-C migrating away from immune synapses into NK cells. Unlike clustering of HLA-C at the immune synapse, intercellular transfer of HLA-C is dependent on NK cell ATP, but not target cell ATP. However, the intercellular transfer of HLA-C is not dependent on active polymerization of the actin cytoskeleton. In addition, different arrangements of HLA-C are seen at inhibitory NK immune synapses, and these alter as NK synapses mature, but in a fashion distinct from that seen upon T cell activation.

The transmembrane sequence of human histocompatibility leukocyte antigen (HLA)-C as a determinant in inhibition of a subset of natural killer cells.

Molecular interactions with the extracellular domains of class I major histocompatibility complex proteins are major determinants of immune recognition that have been extensively studied both physically and biochemically. However, no immunological function has yet been placed on the transmembrane or cytoplasmic amino acid sequences of these proteins despite strict conservation of unique features within each class I major histocompatibility complex locus. Here we report that lysis by a subset of natural killer (NK) cells inhibited by target cell expression of human histocompatibility leukocyte antigen (HLA)-Cw6 or -Cw7 was not inhibited by expression of chimeric proteins consisting of the extracellular domains of HLA-C and the COOH-terminal portion of HLA-G. Assays using transfectants expressing a variety of HLA-Cw6 mutants identified the transmembrane sequence and, in particular, cysteine at position 309 as necessary for inhibition of 68% (25/37) of NK cell lines and 23% (33/145) of NK clones tested. Moreover, these NK clones inhibited by target cell expression of HLA-Cw6 and dependent upon the transmembrane sequence were found not to express or to only dimly express NK inhibitory receptors (NKIR1) that are EB6/HP3E4-positive. Furthermore, assays using monoclonal antibody blocking suggest that an NK receptor other than NKIR1 or CD94 is responsible for recognition dependent upon the transmembrane sequence of HLA-Cw6.

An improved strip FRAP method for estimating diffusion coefficients: correcting for the degree of photobleaching.

Fluorescence recovery after photobleaching is a widely established method for the estimation of diffusion coefficients, strip bleaching with an associated recovery curve analysis being one of the simplest techniques. However, its implementation requires near 100% bleaching in the region of interest with negligible fluorescence loss outside, both constraints being hard to achieve concomitantly for fast diffusing molecules. We demonstrate that when these requirements are not met there is an error in the estimation of the diffusion coefficient D, either an under- or overestimation depending on which assumption is violated the most. We propose a simple modification to the recovery curve analysis incorporating the concept of the relative bleached mass m giving a revised recovery time parametrization tau=m(2)w(2)/4piD for a strip of width w. This modified model removes the requirement of 100% bleaching in the region of interest and allows for limited diffusion of the fluorophore during bleaching. We validate our method by estimating the (volume) diffusion coefficient of FITC-labelled IgG in 60% glycerol solution, D= 4.09 +/- 0.21 microm(2) s(-1), and the (surface) diffusion coefficient of a green-fluorescent protein-tagged class I MHC protein expressed at the surface of a human B cell line, D= 0.32 +/- 0.03 microm(2) s(-1) for a population of cells.

Fourier-transformed infrared photoacoustic spectroscopy of biological materials.

A new technique for measuring the infrared spectra of solids has been developed. The photoacoustic spectra of hemin, hemoglobin, protoporphyrin IX, and horseradish peroxidase show how this technique can be used to obtain structural information about biological materials which cannot readily be studied by normal transmission infrared spectroscopy. The method requires milligram quantities of material and involves no sample preparation.

Protection from natural killer cell-mediated lysis by HLA-G expression on target cells.

The outermost layer of the human placenta is devoid of classical class I human leukocyte antigens (HLA-A, HLA-B, and HLA-C) and class II proteins (HLA-DR, HLA-DQ, and HLA-DP). Although this prevents recognition by maternal T lymphocytes, the lack of class I molecules leaves these cells susceptible to attack by natural killer (NK) cells. However, trophoblast cells directly in contact with the maternal tissues express the class I molecule HLA-G, which may be involved in protecting the trophoblast from recognition by NK cells. Here evidence is provided that expression of HLA-G is sufficient to protect otherwise susceptible target cells from lysis by activated NK1 and NK2 cell lines and clones that are specific for distinct groups of HLA-C alleles. The receptors on NK cells that recognize HLA-G are also identified.

Enhancement of class II-restricted T cell responses by costimulatory NK receptors for class I MHC proteins.

An important feature of the human immune system is the ability of T cells to respond to small quantities of antigen. Class II major histocompatibility complex (MHC)-restricted T cells that expressed a costimulatory natural killer (NK) cell receptor for class I MHC proteins were cloned. In the presence of low doses of superantigen, the proliferative response of these T cell clones was three- to ninefold greater when the T cells were costimulated by way of the NK receptor. Thus, the action of costimulatory NK receptors on T cells may play a significant role in initiating and sustaining immune responses.

Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging.

We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.

Evaluation of nocturnal penile tumescence in the differential diagnosis of sexual impotence. A quantitative study.

Nocturnal penile tumescence (NPT) was assessed in 30 impotent patients for the purpose of differential diagnosis. In 11 psychogenic cases, with several exceptions, NPT was found to be normal, with a marked discrepancy between the NPT findings and the patients' estimates of their daytime erectile capacity. In 19 organic patients, maximal NPT corresponded closely to and mirrored the patient's subjective estimate of his impaired waking performance. Statistically, the figures for frequency, degree, duration, and amount of NPT were found to be significantly and markedly greater in the psychogenic group. Statistical significance was maintained when the 11 psychogenic patients were closely age-matched with 11 of the 19 organic cases. The method is not 100% accurate; some 15% of impotent patients without organic findings have reduced NPT, several of whom were shown to be psychogenic, but others in this group may have undetected organic pathology. The NPT method is very promising, but beset by a number of methodological problems.

Childhood precursors of schizophrenia: facial expressions of emotion.

OBJECTIVE: This study sought to determine whether affective abnormalities could be detected in home movies of children who later developed schizophrenia. METHOD: Trained observers coded facial expressions of emotion of 32 schizophrenic patients and 31 of their healthy siblings from home movies made when they were children. All of the patients met the DSM-III criteria for schizophrenia with onset in late adolescence or early adulthood. The sibling comparison subjects had no history of psychiatric illness. Nine emotions and a neutral category were rated. RESULTS: Analyses revealed significantly lower proportions of joy expressions among the total expressions of the preschizophrenic female subjects than among the same-sex healthy siblings. This difference extended from infancy through adolescence. Among the male subjects, there were inconsistent differences between diagnostic groups in expressions of joy across age levels. However, both the preschizophrenic male subjects and the preschizophrenic female subjects showed greater negative affect than their same-sex comparison groups. CONCLUSIONS: These findings lend support to the assumption that vulnerability to schizophrenia may be subtly manifested in emotional behavior long before the onset of clinical symptoms.

N-linked carbohydrate on human leukocyte antigen-C and recognition by natural killer cell inhibitory receptors.

The possible role of carbohydrate in the interaction of HLA-C with a human inhibitory natural Killer cell Immunoglobulin-like Receptor with two Ig domains, KIR2DL1, was investigated. Transfectants of 721.221 (a class I MHC-negative human B cell line) expressing only HLA-Cw4 or -Cw6 or their respective non-glycosylated mutants (N86Q, S88A) were made. The binding of a KIR2DL1-Ig fusion protein to the non-glycosylated mutant HLA-Cw4- or -Cw6-expressing cells was markedly decreased compared to the wild type-expressing cells. The ability to induce an inhibitory signal in the NK tumor line YTS transfected with KIR2DL1 was also impaired in the nonglycosylated mutant expressing cells. Furthermore, in a second functional assay, mutant HLA-Cw4 and -Cw6 molecules had impaired ability to induce signal transduction in BW cells expressing a KIR2DL1-CD3 zeta chain chimeric protein. Thus, the deletion of the N-linked glycosylation signal in HLA-Cw4 and -Cw6 greatly reduced recognition by KIR2DL1. Alternative interpretations of the data are discussed.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Successful oesophageal pH monitoring with a catheter-free system.

BACKGROUND: Traditional catheter-based oesophageal pH testing is limited by patient discomfort and the tendency for patients to alter their diet and activities during the study. A catheter-free pH monitoring system (Bravo) designed to avoid these problems has recently become available, but the advantages and limitations of this device have not been fully explored. AIM: To report our initial experience with this new technology. METHODS: The records of consecutive patients undergoing Bravo pH monitoring were reviewed. The squamo-columnar junction was localized endoscopically and the pH capsule was placed 6 cm above this junction. All patients were re-endoscoped immediately following placement to document mucosal attachment. Patients were monitored for 24-48 h and then returned the radiotelemetry recording device. Data were subsequently downloaded to a personal computer. RESULTS: Sixty studies were performed over an 11-month period. In seven of the 60 (12%), the probe did not attach properly, but in six of these a replacement probe was prepared and deployed without difficulty. In one case, the probe could not be attached after two attempts and the procedure was abandoned. During one procedure, the probe was attached to the mucosa at a point 9 cm from the squamo-columnar junction, but a positive test result was obtained. In two cases, the data were not initially retrievable from the recorder, but in one case the manufacturer was able to retrieve the data overnight. Finally, two patients were away from the data recorder for extended periods, resulting in a loss of data, in two cases, but there was sufficient information for interpretation in both studies. Therefore, adequate diagnostic data were obtained in 58 of the 60 (97%) studies. CONCLUSIONS: Catheter-free pH testing is a major advance in patient convenience and comfort. The technical difficulties associated with this new technology are minimal and appear to be no more frequent than those seen with catheter-based systems.

Comparative safety of two recombinant hepatitis B vaccines in children: data from the Vaccine Adverse Event Reporting System (VAERS) and Vaccine Safety Datalink (VSD).

BACKGROUND: Preliminary review of data from the Vaccine Adverse Event Reporting System (VAERS), 1991-1994, revealed that more serious adverse events were reported in children who received a specific brand of recombinant hepatitis B (HepB) vaccine. OBJECTIVE: To compare the post-marketing safety experience of the two recombinant HepB vaccines licensed for use in infants and children in the United States. DESIGN: Review of a case series derived from passive surveillance data in the national VAERS. A retrospective cohort study using data from one health maintenance organization participating in Vaccine Safety Datalink (VSD), a computerized record linkage system. POPULATIONS STUDIED: U.S. children, ages birth-10 years for whom adverse events after HepB vaccine were reported to VAERS, 1991-1994. Children, ages birth-6 years, who received HepB vaccine at Kaiser Permanente Medical Care Program, Northern California, 1991-1994. MAIN OUTCOME MEASURES: VAERS reporting rates for each vaccine by manufacturer were calculated from the numbers of reported events occurring within 30 days of HepB vaccination and the number of doses distributed by the manufacturers. VSD event rates for each vaccine were calculated from the numbers of hospitalization or emergency room visits within 30 days of HepB vaccination and the number of vaccine doses administered to the cohort. RESULTS: In VAERS, higher rates of serious events (i.e., life threatening or resulting in hospitalization or permanent disability) were reported in children who received Vaccine A vs. Vaccine B (relative risk [RR]: 3.13-8.18, P < 0.01), particularly by those vaccinated in the private (RR: 7.62-28.58, P < 0.01), but not public sector (RR: 2.12, P = 0.19). Similar types of events were reported in recipients of both vaccines. In contrast, analysis of VSD data showed no significant difference in rates of hospitalization or ER visits in children who received either HepB vaccine (RR: 0.96-1.25, P > 0.05). CONCLUSIONS: Our investigation reveals that it is unlikely there is a true difference between rates of serious events temporally associated with the two HepB vaccines in children. This study demonstrates the dual roles played by VAERS and VSD in providing a more complete picture of the post-marketing safety profile of childhood vaccines, and underscores the importance of using other analytic studies to evaluate findings from passive surveillance systems of adverse events.

Recombinant hepatitis B vaccination of neonates and infants: emerging safety data from the Vaccine Adverse Event Reporting System.

OBJECTIVES: To evaluate the postmarketing safety of recombinant hepatitis B (HB) vaccine given to neonates and infants in the US. METHODS: US reports associated with HB vaccination and received between January 1, 1991, and May 31, 1995, by the national Vaccine Adverse Events Reporting System (VAERS) were reviewed as a case series. RESULTS: During 1991 through 1994, 12,520 (32%) VAERS reports were received for events temporally associated with administration of HB vaccine, of which 14% were received for neonates and infants. More reports described serious outcomes for neonates (< 0.1 year old) than for other age groups (40% vs. 6 to 15%). HB alone was administered to 58 (97%) neonates; review of these reports did not reveal unexpected serious events. Among infants (0.1 to 0.9 years old) 192 (9%) received HB vaccine alone and 1469 (66%) received HB in combination with diphtheria-tetanus-pertussis (DTP) vaccine. Similar serious adverse events reported in neonates and infants included fever, agitation and apnea. Events reported for infants receiving HB/DTP and DTP alone were similar and differed from reports filed for infants receiving HB vaccine alone, suggesting that these events may be associated with use of DTP vaccine. CONCLUSIONS: This review shows no unexpected adverse events in neonates and infants given HB vaccine despite use of at least 12 million doses of vaccine given in these age groups. Although VAERS lacks the ability to distinguish coincidental events from true vaccine reactions, this database represents the largest case series of events temporally associated with HB vaccination of neonates and infants.

Pharmacokinetic study of indicine N-oxide in pediatric cancer patients.

Pharmacokinetics of the experimental antitumor agent indicine N-oxide were investigated in a group of 23 pediatric cancer patients. Plasma elimination of indicine N-oxide was best described by a two-compartment open model. The mean plasma distribution phase half-life, plasma elimination phase half-life, and plasma clearance were 8 min, 84 min, and 62 ml/min/m2 (2.1 ml/min/kg), respectively. One patient with renal impairment had an abnormally long plasma elimination phase half-life (275 min) and reduced plasma clearance (17 ml/min/m2). Plasma elimination phase half-life values increased and plasma clearance values decreased with increasing age of the pediatric patients. Plasma elimination of indicine N-oxide was more rapid in this group of children than in adults who had previously received the drug.

Fractography of a bis-GMA resin.

The fracture behavior of a bis-GMA resin was studied by means of the double-torsion test. The fracture parameter measured was the stress-intensity factor. Fracture occurred in either a stick-slip (unstable) or continuous (stable) manner, depending upon the test conditions. When stick-slip propagation occurred, the fracture surfaces showed characteristic crack-arrest lines. The fracture surfaces were examined by use of a reflected-light optical microscope. The stress-intensity factor for crack initiation was found to be related to the size of the crack-arrest line which, in turn, could be related to the Dugdale model for plastic zone size. The evidence supported the concept that the behavior of the crack during propagation was controlled by the amount of plastic deformation occurring at the crack tip.

An investigation into the fracture behavior of a particulate-filled bis-GMA resin.

Dental composites are being increasingly used to restore occlusal surfaces of posterior teeth. If these restorations are not to fracture in service, an understanding of their fracture behavior is essential. The fracture parameter used is the stress-intensity factor at crack instability (KIC). This has been calculated by means of the double-torsion test in a mechanical testing machine. The fracture behavior of the composite was studied by varying: the amount of filler (7%, 15%, 26%, and 41% by volume); the surface treatment of the filler (coated or uncoated); the environment (air and water); and the cross-head rate (0.05, 0.5, 5, and 50 mm/min). Fracture was found to occur in either a continuous (stable) or stick-slip (unstable) manner. The stick-slip behavior was due to blunting of the crack tip, which was controlled by the yield behavior. When there was no significant blunting, continuous crack growth occurred. An unique relationship was found to exist between the yield stress and the stress-intensity factor for all conditions of testing.

The fluorescence and circular dichroism of proteins in reverse micelles: application to the photophysics of human serum albumin and N-acetyl-L-tryptophanamide.

Evidence is presented that a compartmentalised protein exists in its native state only within a particular size of aqueous cavity. This behaviour is shown to exist in AOT reverse micelles using fluorescence quenching and circular dichroism (CD) studies of human serum albumin (HSA). In particular, far ultraviolet CD measurements show that a reduction in quencher accessibility to the fluorophore is consistent with the protein being nearest to its native conformation at a waterpool size of around 80 A diameter. We also show that the biexponential fluorescence decay of N-acetyl-L-tryptophanamide (NATA) in AOT reverse micelles arises from the probe being located in two distinct sites within the interfacial region. The more viscous of these two sites is located on the waterpool side of the interface and the other is located on the oil side of the interface.

Weight of evidence supports one operation for "ratios" and "differences" of heaviness.

This paper investigates an apparent contradiction between recent studies of "ratios" and "differences" of heaviness. Birnbaum and Veit (1974) found a single rank order for judgments in the two tasks, whereas Rule, Curtis, and Mullin (1981), who used a different stimulus set, procedure, and experimental design, reported two orders. To investigate the cause of this discrepancy, the present study manipulated the experimental design using the same stimuli and procedure as Rule et al. (1981). In one experiment (within-subject designs), each subject judged all combinations of the standard and comparison stimulus; in the other experiment (between-subjects designs) each subject received only one standard, and different groups of subjects were given different standards. "Ratios" and "differences" of heaviness were monotonically related for the majority of subjects who judged all combinations of standards and comparisons. Variations in the modulus and response examples did not affect the rank order of "ratios" within subjects. These results suggest that the contradiction in results is due to the difference in experimental design rather than differences in stimuli or procedure. In the between-subjects designs, the rank order of the "ratio" judgments depended on the standards and examples. Both previous and present results are consistent with the theory that subjects use one operation, subtraction, for both tasks and that the judgment function varies with between-subjects manipulations of the standard, examples, and modulus.