Elevated baseline soluble FcεRI may be linked to early response to omalizumab treatment in chronic spontaneous urticaria.

BACKGROUND: Omalizumab, an anti-IgE monoclonal antibody, is an effective treatment in chronic spontaneous urticaria (CSU). Predictors of fast and good response for omalizumab treatment have not yet been identified and characterized. OBJECTIVE: To evaluate whether soluble FcεRI (sFcεRI), a marker of IgE-mediated mast cell activation, predicts the time of response to omalizumab in CSU. METHODS: Sera of 67 CSU patients were obtained before omalizumab treatment and analysed for sFcεRI levels by ELISA (2 ng/mL was used as cut-off for elevated sFcɛRI). Treatment response during the first 4 weeks was assessed with the urticaria activity score (UAS7), urticaria control test (UCT) and the rolling UAS7 (rUAS7). RESULTS: Elevated pre-treatment sFcɛRI levels were detected in more than 70% of patients with completely controlled disease (UCT = 16) and well-controlled disease (UCT = 12-15) and were significantly associated with disease control (χ2 = 4.94, p < 0.05). More than half of the patients (14/25) with low levels had poor disease control (UCT < 12). Of the patients who achieved complete and marked UAS7 response, respectively, 75% and 63% had elevated baseline sFcɛRI levels. Post-treatment UAS7 scores were lower in patients with elevated sFcɛRI levels reaching statistical significance at Week 3 (p < 0.05). Patients with elevated baseline sFcɛRI levels achieved rUAS7 ≤ 6 and = 0 earlier than those with lower levels (Days 9 vs. 13 and Days 12 vs. 14, respectively). CONCLUSION: Elevated sFcεRI serum levels predict early and good response to treatment with omalizumab, which may help to better design treatment options for CSU patients.

Autologous serum skin test reactions in chronic spontaneous urticaria differ from heterologous cell reactions.

BACKGROUND: Autoimmune chronic spontaneous urticaria (CSU) is due to mast cell (MC)-activating autoantibodies, which are screened for by the autologous serum skin test (ASST) and basophil tests (BTs). Many CSU patients are positive in only one of these tests. How often this occurs and why is currently unknown. OBJECTIVES: To characterize the prevalence of mismatched ASST and BTs in CSU patients, and to investigate possible reasons for these mismatches. METHODS: We determined the rates of ASST+/BT- and ASST-/BT+ mismatches in published CSU studies. We assessed sera from 48 CSU patients by ASST, two BTs (basophil histamine release assay, BHRA; basophil activation test, BAT), a MC histamine release assay (MCHRA) and by ex vivo skin microdialysis (SMD). RESULTS: The ASST/BT mismatch rate in published CSU studies was 31% (ASST+/BT-: 22%, ASST-/BT+: 9%). In our patients, the ASST/BHRA and ASST/BAT mismatch rate was 35.4% (ASST+/BHRA-: 18.8% and ASST-/BHRA+: 16.7%) and 31.3% (ASST+/BAT-: 6.3% and ASST-/BAT+: 25.0%), respectively, and the two BTs were significantly correlated (P = 0.0002). The use of heterologous MCs, in vitro and in situ, instead of basophils produced similar results (MCHRA mismatch: 47.9%, ASST+/MCHRA-: 18.8%, ASST-/MCHRA+: 29.2%; SMD mismatch: 40.0%, ASST+/SMD-: 10.0% and ASST-/SMD+: 30.0%), and the MCHRA was highly correlated with SMD results (P = 0.0002). CONCLUSIONS: The ASST and BTs show divergent results in a third of CSU patients. Mismatches cannot be explained by the choice of basophil assay, the type of heterologous cells exposed to CSU serum in vitro (basophils vs. mast cells), nor the experimental setting of heterologous skin mast cells (in vitro vs. in situ). Thus, serum-induced whealing, in CSU patients, seems to involve autologous skin signals modulating MC degranulation.

Immunological effects and potential mechanisms of action of autologous serum therapy in chronic spontaneous urticaria.

BACKGROUND: Autoimmune processes are considered to play a major role in the pathogenesis of chronic spontaneous urticaria (CSU). Very recently, interleukin 24 (IL-24) has been identified as an immunoglobulin E (IgE) autoantigen in CSU. Some studies revealed that notably autologous serum skin test (ASST)-positive CSU patients may benefit from autohemotherapy; however, the mechanisms of action remain unknown. We aimed to investigate the immunological effects of autologous serum injections in ASST-positive CSU patients. METHODS: Sixty-six ASST-positive CSU patients were treated with weekly intramuscular autologous serum injections for 8 weeks and followed up for 12 weeks. Urticaria activity score (UAS7) and Dermatology Life Quality Index (DLQI) were assessed. The ASST was done at baseline, week 9 and week 21. Serum samples (baseline, weeks 9, 13 and/or 21) were analysed for the levels of IgE-anti-IL-24 and immunoglobulin G (IgG)-anti-IL-24 via ELISA and their ability to release histamine in basophils [basophil histamine release assay (BHRA)]. RESULTS: Autologous serum therapy resulted in a substantial improvement in disease activity and quality of life after 8 and 20 weeks. Twenty-eight percent and 34% of patients turned ASST-negative in weeks 9 and 21, respectively, but there was no link between their response to treatment and changes of ASST results. Also, no significant or relevant changes in BHRA were observed. In contrast, autologous serum therapy significantly decreased IgE-anti-IL-24 serum levels, but not IgG-anti-IL-24 serum levels, in responders but not in non-responders. CONCLUSIONS: Our findings suggest that the immunological effects of autologous serum therapy include a reduction in IgE-anti-IL24 autoantibodies, which may contribute to the pathogenesis of CSU.

Spleen tyrosine kinase inhibition blocks airway constriction and protects from Th2-induced airway inflammation and remodeling.

BACKGROUND: Spleen tyrosine kinase (Syk) is an intracellular nonreceptor tyrosine kinase, which has been implicated as central immune modulator promoting allergic airway inflammation. Syk inhibition has been proposed as a new therapeutic approach in asthma. However, the direct effects of Syk inhibition on airway constriction independent of allergen sensitization remain elusive. METHODS: Spectral confocal microscopy of human and murine lung tissue was performed to localize Syk expression. The effects of prophylactic or therapeutic Syk inhibition on allergic airway inflammation, hyperresponsiveness, and airway remodeling were analyzed in allergen-sensitized and airway-challenged mice. The effects of Syk inhibitors BAY 61-3606 or BI 1002494 on airway function were investigated in isolated lungs of wild-type, PKCα-deficient, mast cell-deficient, or eNOS-deficient mice. RESULTS: Spleen tyrosine kinase expression was found in human and murine airway smooth muscle cells. Syk inhibition reduced allergic airway inflammation, airway hyperresponsiveness, and pulmonary collagen deposition. In naïve mice, Syk inhibition diminished airway responsiveness independently of mast cells, or PKCα or eNOS expression and rapidly reversed established bronchoconstriction independently of NO. Simultaneous inhibition of Syk and PKC revealed additive dilatory effects, whereas combined inhibition of Syk and rho kinase or Syk and p38 MAPK did not cause additive bronchodilation. CONCLUSIONS: Spleen tyrosine kinase inhibition directly attenuates airway smooth muscle cell contraction independent of its protective immunomodulatory effects on allergic airway inflammation, hyperresponsiveness, and airway remodeling. Syk mediates bronchoconstriction in a NO-independent manner, presumably via rho kinase and p38 MAPK, and Syk inhibition might present a promising therapeutic approach in chronic asthma as well as acute asthma attacks.

Determination of sIgE to rPhl p 1 is sufficient to diagnose grass pollen allergy.

BACKGROUND: New diagnostic tools such as the basophil activation test (BAT) and component-resolved diagnosis (CRD) are promising for Hymenoptera venom or food allergy. A clear benefit for inhalant allergens has not yet been shown. Our aim was to compare new and established tests for grass pollen allergy. METHODS: Forty-nine patients with grass pollen allergy and 47 controls were prospectively enrolled in the study. A symptom score was calculated for each patient. Conjunctival provocation tests (CPT), skin prick tests (SPT), BAT, and sIgE determination including CRD were performed. Sensitivity and specificity were compared and results were correlated with the symptom score. RESULTS: Single determination of sIgE to rPhl p 1 showed the best balance between sensitivity (98%) and specificity (92%). Use of additional components, such as rPhl p 2 and 5, did not increase sensitivity. Generally, sensitivity of tests was high: SPT 100%, ISAC-112 100%, sIgE to timothy grass 98%, BAT 98%, ISAC-103 84%, and CPT 83%. Specificity ranged from 79% (SPT) to 96% (CPT). All test results and calculated values (e.g. ratio sIgE/tIgE) did not correlate with symptom severity. Asymptomatic sensitization to timothy grass in controls was rare in the CAP (11%) and predominantly due to Phl p 1 sensitization. CONCLUSION: rPhl p 1 was sufficient to diagnose grass pollen allergy, and sIgE patterns were the same in symptomatically and asymptomatically sensitized subjects. The testing of multiple components was of minor importance, and no test correlated with symptom severity.

Aspects of static and dynamic motor function in peripheral nerve regeneration: SSI and CatWalk gait analysis.

Assessment of the therapeutic potential of interventions to bridge-repair peripheral nerve defects heavily relies on the demonstration of improved functional outcome. In the present study we used CatWalk gait analysis (locomotor-test) and Static Sciatic Index (SSI) (static-toe-spread-test) to assess the behavioural benefits of autologous nerve transplantation (ANT) repair of 2-cm rat sciatic nerve defects (neurotmesis-lesion). A reproducible and standardised rat sciatic nerve crush lesion model (axonotmesis-lesion) was used to assess the extent of recovery supported by maximal axon regeneration (measured by SSI and CatWalk). Animals were behaviourally followed for a period of 10 weeks. SSI analysis showed that ANT induced a significant improvement in motor deficit from about -95 to -65, however, CatWalk analysis did not show any major indication of locomotor recovery. This discrepancy might suggest that improvements in static motor functions (such as toe spreading) could reflect an early indicator for the recovery of function. We also noted differences in axon regeneration including increased axon density, smaller axon diameters and thinner myelin sheaths in the distal region of the ANT in comparison to the equivalent region of crushed and normal nerves. This difference in axon regeneration may be related to the clearly improved toe spreading function. We conclude that SSI and CatWalk present different advantages and disadvantages for the assessment of motor recovery after bridge-repair of peripheral nerve defects.

CatWalk gait analysis in assessment of functional recovery after sciatic nerve injury.

Following peripheral nerve injury repair, improved behavioural outcome may be the most important evidence of functionality of axon regeneration after any repair strategy. A range of behavioural testing paradigms have been developed for peripheral nerve injury research. Complete injury of the adult rat sciatic nerve is frequently used in combination with walking track analysis. Despite its wide-spread use, these walking track analyses are unsuitable for the simultaneous assessment of both dynamic and static gait parameters. Conversely, a novel automated gait analysis system, i.e. CatWalk can simultaneously measure dynamic as well as static gait parameters and, importantly, it's easy to control for the speed of locomotion which can strongly affect gait parameters. In a previous study, CatWalk was already successfully used to examine deficits in both dynamic and static gait parameters using the sciatic nerve lesion model with a 1cm gap characterized by absence of recovery [Deumens R, Jaken RJ, Marcus MA, Joosten EA. The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection. J Neurosci Methods 2007;164:120-30]. Using the sciatic nerve crush injury model (validated with the static sciatic index) and a follow-up period of 12 weeks, we now show that CatWalk can also measure behavioural recovery. In particular dynamic gait parameters, coordination measures, and the intensity of paw prints are of interest in detecting recovery as far as these parameters completely return to pre-operative values after crush injury. We conclude that CatWalk can be used as a complementary approach to other behavioural testing paradigms to assess clinically relevant behavioural benefits, with a main advantage that CatWalk demonstrates both static and dynamic gait parameters at the same time.

Confinement scaling laws for the conventional reversed-field pinch

A series of high resolution, 3D, resistive MHD numerical simulations of the reversed-field pinch are performed to obtain scaling laws for poloidal beta and energy confinement at Lundquist numbers approaching 10(6). Optimum plasma conditions are attained by taking the transport coefficients to be classical, and ignoring radiation losses and resistive wall effects. We find that poloidal beta scales as beta(straight theta) approximately I-0.40 and that the energy confinement time scales as tau(E) approximately I0.34 for fixed I/N, with aspect ratio R/a = 1.25.

Persistence of hepatitis C virus in a human megakaryoblastic leukaemia cell line.

Thrombocytopenia is a frequent clinical finding in patients with hepatitis C virus (HCV) infection. Platelets from patients with HCV infection have been identified as carriers of HCV RNA in our previous studies. The present study was designed to further investigate the possibility of HCV replication in megakaryoblasts from which platelets are eventually released. A megakaryoblastic cell line (MEG-01), established from a chronic myelogenous leukaemia patient 13 years ago, was used for this study. The MEG-01 cells were inoculated with fresh serum from a patient with HCV infection and renamed MEG-01-I cells. Surprisingly, both MEG-01 and MEG-01-I were positive by HCV reverse transcription-polymerase chain reaction (RT-PCR) for the existence of HCV RNA and minus-strand HCV RNA, regardless of inoculation. This was further confirmed by in situ RT-PCR. The HCV antigens, such as core, envelope, and non-structural (NS)3 and NS4, were also present in both cell lines, as identified by Western blotting and indirect immunofluorescence staining. In addition, virus-like particles were observed by electron microscopy in the MEG-01 cell line as well as in the MEG-01-I cell line. These findings indicate that the megakaryoblasts are vulnerable to HCV infection and that replication of HCV can occur in these cells. This may help us to better understand the pathogenesis of thrombocytopenia in patients with HCV infection. The MEG-01 cell line, which may have been continuously shedding HCV for years, should be a useful model for experimental research into HCV.

Significance of the anti-E2 response in self-limited and chronic hepatitis C virus infections in chimpanzees and in humans.

To determine whether there was a correlation between the kinetics or frequency of antibody to mammalian-derived hepatitis C virus (HCV) second envelope protein (E2) and development of chronicity or self-limitation of HCV infections, serial sera were examined for anti-E2, anti-HCV with confirmation with Matrix 2.0 (Abbott Laboratories, Abbott Park, IL), and reverse transcriptase-polymerase chain reaction (RT-PCR) from 6 cases of self-limited infection and 6 cases of chronic infection in chimpanzees, and from 5 cases of self-limited infection and 3 cases of chronic infection in patients. Anti-E2 developed earlier, more frequently, and to higher titer in chimpanzees and patients who were developing chronic infection than in those with self-limited infections. Thus anti-E2 is unlikely to play a role in self-limitation of the infection. However, long-term persistence of anti-E2 correlates with chronic infection. There was little or no correlation between the timing of development of anti-E2 and anti-HCV.

Monoclonal antibodies to an HIV-1 group O envelope recombinant.

Monoclonal antibodies were developed to a recombinant HIV-I group O envelope protein derived from the isolate HAM112. These monoclonal antibodies were characterized for reactivity to a series of overlapping synthetic peptides (29-30 mers) covering gp120 C-terminal and gp41 ectodomain regions of the HIV-1 group O envelope protein. Most of these monoclonal antibodies reacted with peptides spanning sequences analogous to HIV-1 group M epitopes identified from studies in mice and humans. However, several of the antibodies that were nonreactive to individual peptides did react to a mixture of longer peptides from the N-terminal and C-terminal helical regions of the gp41 ectodomain. The monoclonal antibodies described in this study are valuable tools for characterization of antigenic differences between HIV-1 group O and group M viruses.

Lack of evidence of hepatitis C infection in 290 blood component recipients, demonstrated by several single-antigen research immunoassays.

BACKGROUND: A group of 290 transfusion recipients enrolled in a prospective study of posttransfusion hepatitis was studied to determine the possibility of previously unrecognized hepatitis C virus (HCV) transmission. STUDY DESIGN AND METHODS: Before and after transfusion, blood specimens that were negative in first-generation enzyme immunoassay (EIA) were tested by current commercial EIAs, several single-antigen research EIAs, and supplemental tests. RESULTS: Current second- and third-generation EIAs identified five subjects (1.7% of total) who had chronic hepatitis C before transfusion. Twenty additional sera had some reactivity with research EIAs. However, those results were the same before and after transfusion (n = 7), had reverted to partially reactive or nonreactive (n = 8), or could not be confirmed by serologic tests or polymerase chain reaction in follow-up specimens (n = 5). CONCLUSIONS: Transient or restricted reactivity to HCV antigens measured by more sensitive research EIAs does not seem to correspond to recent HCV transmission by transfusion. Whether such reactivity could reflect remote HCV infection, with the potential for chronic or intermittent viremia, remains to be determined.

Hepatitis C virus antibodies in systemic lupus erythematosus.

To determine the prevalence and significance of serum antibody to hepatitis C virus (HCV) in patients with systemic lupus erythematosus (SLE), we measured serum antibodies to HCV by enzyme-linked immunosorbent (ELISA) and by Abbott MATRIX Immunoblot assays in 42 patients with SLE, a condition associated with hypergammaglobulinemia. We used the polymerase chain reaction (PCR) to identify patients with HCV viremia. Five of 42 (11.9%) patients were seropositive for anti-HCV by ELISA; of these only two were positive by PCR; only one of three patients seropositive by Immunoblot assay was also positive by PCR. Both ELISA and the Immunoblot assays may be falsely positive for ongoing HCV infection in patients with SLE. Suspected HCV infection should be confirmed with PCR for serum HCV RNA in these patients.

Acute sporadic non-A, non-B, non-C, non-D, non-D, non-E hepatitis.

Patients presenting with clinical and laboratory features consistent with a diagnosis of acute non-A, non-B hepatitis were evaluated for evidence of hepatitis C or hepatitis E infection and for evidence of severe or prolonged disease. Antibody to hepatitis C virus (anti-HCV) was detected in 75 of 108 (69%) patients, antibody to hepatitis E virus (anti-HEV) in three patients (3%), and neither antibody in 31 (29%) patients. One patient had both anti-HCV and anti-HEV. HCV RNA was not detected in sera from any of 20 patients with seronegative (non-ABCDE) hepatitis, but in all 10 patients with anti-HCV who were tested by polymerase chain reaction (PCR). Compared with patients with acute hepatitis C, those with non-ABCDE hepatitis had a lower incidence of parenteral risk factors (6% vs. 70%; P < .001), higher peak serum bilirubin levels (45% vs. 5% with peak levels > 15 mg/dL; P < .001), more prolonged jaundice (25% vs. 0% with peak bilirubin >5 weeks after onset; P < .01), more severe prothrombin time abnormalities (26% vs. 0% with >3 second prolongation; P < .001), more severe hypoalbuminemia (39% vs. 9% with albumin <3 g/dL; P < .01), and more frequent major clinical complications (13% vs. 0% with encephalopathy; P < .01; 10% vs. 0% with death or transplant; P = .024). Patients with acute non-ABCDE hepatitis were less likely to develop chronic hepatitis than those with acute hepatitis C (23% vs. 68%; P < .05). Thus, patients with acute non-ABCDE hepatitis are epidemiologically distinct from those with acute hepatitis C and have a significantly more severe acute illness.

A search for hepatitis C virus polymerase chain reaction-positive but seronegative subjects among blood donors with elevated alanine aminotransferase.

BACKGROUND: Previous studies reported the existence of hepatitis C virus (HCV) polymerase chain reaction (PCR)-positive but seronegative sera. This is not surprising in the case of window-phase specimens, because PCR can detect HCV RNA many weeks before the appearance of antibody. To determine whether such sera can also be found in chronically infected subjects, a high-risk population of blood donors with elevated alanine aminotransferase was studied. STUDY DESIGN AND METHODS: Freshly frozen plasma from 301 donors with alanine aminotransferase > 100 IU per L was tested with PCR assays that were rigidly controlled for specificity and contamination, and with current and newer versions of assays for anti-HCV. Sera were classified as seropositive if positive in two screening assays and one supplemental assay or if positive in two screening assays and PCR. RESULTS: New versions of screening assays detected 100 percent of seropositive samples. A second-generation immunoblot assay detected 98 percent of seropositive sera, a second-generation recombinant immunoblot assay detected 96 percent, and an enzyme immunoassay for antibody to the envelope protein of HCV detected 98 percent. Fifty-one of 54 seropositive sera were PCR positive. None of the 247 seronegative samples was reproducibly positive on PCR. CONCLUSION: No PCR-positive but seronegative donors were found in this high-risk donor population. The possible benefit of PCR screening of blood donors can be determined only by large-scale comparative testing of donor populations and may be limited to the detection of window-phase infections.

Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: a new marker of HCV infection closely associated with viremia.

The second envelope protein (E2) of the hepatitis C virus (HCV) was cloned and expressed in Chinese hamster ovary (CHO) cells. This E2 glycoprotein was purified using ion exchange and lectin chromatography and used to construct an enzyme immunoassay for HCV E2 antibodies. The assay was shown to have good specificity, and detection of E2 antibodies was positively correlated (97.3%) to the presence of HCV RNA in serum and plasma. A high concordance between HCV 2.0 and E2 EIA reactivities was also observed. E2 antibody was the first serological marker to appear in 3/5 HCV seroconversion panels. This work demonstrated that 42.4% of core and 15.4% of NS3 indeterminate specimens also contained antibodies to E2, suggesting that HCV infection had occurred in these individuals. The E2 antibody assay was used to evaluate HCV 2.0 EIA-positive, HCV 3.0 EIA-negative plasma donors with indeterminate reactivity on RIBA HCV 2.0 or MATRIX HCV 1.0. Several HCV 3.0-negative specimens were shown to contain E2 antibodies in addition to an original indeterminate serological marker, primarily core. It is concluded that anti-E2 is a useful marker for determining HCV infection, and that the presence of antibodies to two nonoverlapping viral gene products suggests true HCV exposure. New HCV 3.0 blood screening tests should detect HCV 2.0-positive donors who present with an indeterminate pattern by RIBA or MATRIX and who also carry E2 antibodies.

Identification of hepatitis C virus by immunoelectron microscopy.

Sequencing of the hepatitis C virus (HCV) has provided a better understanding of the natural history, immunology, and epidemiology of this virus. However, the morphology of HCV has not been definitively characterized. In this study, through a sequence of concentration processes, virus-like particles were isolated from human serum and liver tissue, visualized by transmission electron microscopy and identified as hepatitis C virion by immunoelectron microscopy. Spherical flavi-like virus particles, approximately 70 nm in diameter, were observed in the fraction with 1.04-1.12 g ml-1 sucrose density and bound to immunogold particles with monoclonal antibodies (mAb) against hepatitis C. The nucleocapsid of the particles, which were 50 nm in diameter, appeared to be icosahedral in structure and surrounded by an envelope covered with surface projections. A 'tadpole' form of particles was also observed. The findings indicate that the low buoyant density in sucrose and the morphological features of the hepatitis C virion are consistent with the characteristics of flaviviruses and pestiviruses.