Increased levels of a pro-inflammatory IgG receptor in the midbrain of people with schizophrenia.

BACKGROUND: There is growing evidence that neuroinflammation may contribute to schizophrenia neuropathology. Elevated pro-inflammatory cytokines are evident in the midbrain from schizophrenia subjects, findings that are driven by a subgroup of patients, characterised as a "high inflammation" biotype. Cytokines trigger the release of antibodies, of which immunoglobulin G (IgG) is the most common. The level and function of IgG is regulated by its transporter (FcGRT) and by pro-inflammatory IgG receptors (including FcGR3A) in balance with the anti-inflammatory IgG receptor FcGR2B. Testing whether abnormalities in IgG activity contribute to the neuroinflammatory abnormalities schizophrenia patients, particularly those with elevated cytokines, may help identify novel treatment targets. METHODS: Post-mortem midbrain tissue from healthy controls and schizophrenia cases (n = 58 total) was used to determine the localisation and abundance of IgG and IgG transporters and receptors in the midbrain of healthy controls and schizophrenia patients. Protein levels of IgG and FcGRT were quantified using western blot, and gene transcript levels of FcGRT, FcGR3A and FcGR2B were assessed using qPCR. The distribution of IgG in the midbrain was assessed using immunohistochemistry and immunofluorescence. Results were compared between diagnostic (schizophrenia vs control) and inflammatory (high vs low inflammation) groups. RESULTS: We found that IgG and FcGRT protein abundance (relative to ß-actin) was unchanged in people with schizophrenia compared with controls irrespective of inflammatory subtype. In contrast, FcGRT and FcGR3A mRNA levels were elevated in the midbrain from "high inflammation" schizophrenia cases (FcGRT; p = 0.02, FcGR3A; p < 0.0001) in comparison to low-inflammation patients and healthy controls, while FcGR2B mRNA levels were unchanged. IgG immunoreactivity was evident in the midbrain, and approximately 24% of all individuals (control subjects and schizophrenia cases) showed diffusion of IgG from blood vessels into the brain. However, the intensity and distribution of IgG was comparable across schizophrenia cases and control subjects. CONCLUSION: These findings suggest that an increase in the pro-inflammatory Fcγ receptor FcGR3A, rather than an overall increase in IgG levels, contribute to midbrain neuroinflammation in schizophrenia patients. However, more precise information about IgG-Fcγ receptor interactions is needed to determine their potential role in schizophrenia neuropathology.

Ambient and laboratory evaluation of a low-cost particulate matter sensor.

Low-cost, light-scattering-based particulate matter (PM) sensors are becoming more widely available and are being increasingly deployed in ambient and indoor environments because of their low cost and ability to provide high spatial and temporal resolution PM information. Researchers have begun to evaluate some of these sensors under laboratory and environmental conditions. In this study, a low-cost, particulate matter sensor (Plantower PMS 1003/3003) used by a community air-quality network is evaluated in a controlled wind-tunnel environment and in the ambient environment during several winter-time, cold-pool events that are associated with high ambient levels of PM. In the wind-tunnel, the PMS sensor performance is compared to two research-grade, light-scattering instruments, and in the ambient tests, the sensor performance is compared to two federal equivalent (one tapered element oscillating microbalance and one beta attenuation monitor) and gravimetric federal reference methods (FEMs/FRMs) as well as one research-grade instrument (GRIMM). The PMS sensor response correlates well with research-grade instruments in the wind-tunnel tests, and its response is linear over the concentration range tested (200-850 µg/m3). In the ambient tests, this PM sensor correlates better with gravimetric methods than previous studies with correlation coefficients of 0.88. However additional measurements under a variety of ambient conditions are needed. Although the PMS sensor correlated as well as the research-grade instrument to the FRM/FEMs in ambient conditions, its response varies with particle properties to a much greater degree than the research-grade instrument. In addition, the PMS sensors overestimate ambient PM concentrations and begin to exhibit a non-linear response when PM2.5 concentrations exceed 40 µg/m3. These results have important implications for communicating results from low-cost sensor networks, and they highlight the importance of using an appropriate correction factor for the target environmental conditions if the user wants to compare the results to FEM/FRMs.

EphA2 promotes infiltrative invasion of glioma stem cells in vivo through cross-talk with Akt and regulates stem cell properties.

Diffuse infiltrative invasion is a major cause for the dismal prognosis of glioblastoma multiforme (GBM), but the underlying mechanisms remain incompletely understood. Using human glioma stem cells (GSCs) that recapitulate the invasive propensity of primary GBM, we find that EphA2 critically regulates GBM invasion in vivo. EphA2 was expressed in all seven GSC lines examined, and overexpression of EphA2 enhanced intracranial invasion. The effects required Akt-mediated phosphorylation of EphA2 on serine 897. In vitro the Akt-EphA2 signaling axis is maintained in the absence of ephrin-A ligands and is disrupted upon ligand stimulation. To test whether ephrin-As in tumor microenvironment can regulate GSC invasion, the newly established Efna1;Efna3;Efna4 triple knockout mice (TKO) were used in an ex vivo brain slice invasion assay. We observed significantly increased GSC invasion through the brain slices of TKO mice relative to wild-type (WT) littermates. Mechanistically EphA2 knockdown suppressed stem cell properties of GSCs, causing diminished self-renewal, reduced stem marker expression and decreased tumorigenicity. In a subset of GSCs, the reduced stem cell properties were associated with lower Sox2 expression. Overexpression of EphA2 promoted stem cell properties in a kinase-independent manner and increased Sox2 expression. Disruption of Akt-EphA2 cross-talk attenuated stem cell marker expression and neurosphere formation while having minimal effects on tumorigenesis. Taken together, the results show that EphA2 endows invasiveness of GSCs in vivo in cooperation with Akt and regulates glioma stem cell properties.

Maternal inheritance of mitochondria in Eucalyptus globulus.

It is important to verify mitochondrial inheritance in plant species in which mitochondrial DNA (mtDNA) will be used as a source of molecular markers. We used a polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) approach to amplify mitochondrial introns from subunits 1, 4, 5, and 7 of NADH dehydrogenase (nad) and cytochrome oxidase subunit II (cox2) in Eucalyptus globulus. PCR fragments were then either sequenced or cut with restriction enzymes to reveal polymorphism. Sequencing cox2 showed that eucalypts lack the intron between exons 1 and 2. One polymorphism was found in intron 2-3 of nad7 following restriction digests with HphI. Fifty-four F1 progeny from seven families with parents distinguishable in their mitochondrial nad7 were screened to show that mitochondria were maternally inherited in E. globulus. These results constitute the first report of mitochondrial inheritance in the family Myrtaceae.

The low-frequency MNS blood group antigens Ny(a) (MNS18) and Os(a) (MNS38) are associated with GPA amino acid substitutions.

BACKGROUND: Antigens of the MNS blood group system are located on two sialoglycoproteins, GPA and GPB, encoded by GYPA and GYPB. The molecular backgrounds of the low-frequency antigens Ny(a) and Os(a) are not known. STUDY DESIGN AND METHODS: Immunoblotting and a monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay were used to analyze Os(a). PCR-amplified products of the coding exons of GYPA were studied by single-strand conformation polymorphism analysis, and exon 3 was sequenced. Synthetic peptides were used in hemagglutination-inhibition tests. RESULTS: Sequencing of GYPA exon 3 of two unrelated Ny(a+) persons revealed heterozygosity for a T194A base change encoding an Asp27Glu substitution. Immunoblotting with anti-Os(a) and an MAIEA assay with MoAbs to GPA showed that Os(a) is on GPA. Sequencing exon 3 of an Os(a+) person from the only family with Os(a) revealed heterozygosity for a C273T base change encoding a Pro54Ser substitution. A synthetic peptide representing part of GPA with the Os(a) mutation (VRTVYPSEEETGE) completely inhibited anti-Os(a), whereas the control peptide (VRTVYPPEEETGE) did not inhibit anti-Os(a). CONCLUSION: Ny(a) and Os(a) are low-frequency antigens of the MNS blood group system that represent Asp27Glu and Pro54Ser substitutions in GPA, respectively.

Validity and reliability of a modified qualitative dietary fat index in low-income, overweight, African American adolescent girls.

This study evaluated the validity and reliability of a modified qualitative dietary fat index questionnaire (QFQ) in an adolescent minority population. The QFQ was administered to study participants twice over a 2-week period, and data were compared with mean values from three 24-hour recalls. Fifty-seven low-income, overweight, African American adolescent girls, aged 11 to 17 years, were recruited from 7 public housing developments in Atlanta, Georgia. To determine validity, the total QFQ score was compared with the mean values of total fat, percentage of energy from fat, and total energy from three 24-hour recalls within 2 weeks of first administration of the QFQ. Reliability was tested in a subsample (n = 22) by comparing total QFQ scores administered 2 weeks apart. Total fat was significantly correlated (r = 0.31, P < .05) with the QFQ score. Total energy (r = 20.23) and percentage of energy from fat (r = -0.23) were not significantly correlated with the QFQ score. The test-retest QFQ scores were significantly correlated (r = 0.54, P < .01). The data suggest that additional modifications are needed to make the QFQ more appropriate for low-income, over-weight, African American adolescent girls.

The monoclonal antibody-specific immobilization of erythrocyte antigens assay (MAIEA) in the investigation of human red-cell antigens and their associated membrane proteins.

The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) technique is an immunoassay devised primarily for locating blood group antigens on specific red-cell membrane proteins. The assay involves the incubation of intact red cells with two antibodies, one human alloantibody, the other a nonhuman antibody, usually a rodent monoclonal antibody, but polyclonal antibodies of rabbit origin have been utilized. For a positive result, both antibodies must bind to the same membrane protein. The red cells are lysed, the membrane solubilized and the trimolecular complex of two antibodies and membrane protein is captured in a well coated with goat antirodent (or rabbit) immunoglobulin. The immobilized complex is then detected by the use of peroxidase-conjugated goat antihuman (or rodent) immunoglobulin. Negative results, due to mutual blocking between the human and animal antibodies when their epitopes are close together on the same molecule, have permitted a degree of localization of epitopes on some proteins. This has been most effective in the mapping of Cromer blood group system antigens on the complement control protein domains of decay-accelerating factor (DAF, CD55), but has also proved informative in the clustering of antigens on the Lutheran and Kell glycoproteins. MAIEA is an effective tool for the identification of antibodies to Knops-system antigens on complement receptor 1 (CR1, CD35) in immunohaematology reference laboratories. These antibodies are clinically unimportant, but must be identified before they can be ignored for transfusion purposes.

Analysis of Knops blood group antigens on CR1 (CD35) by the MAIEA test and by immunoblotting.

Kna, McCa, Sla and Yka are red cell antigens of relatively high frequency, located on complement receptor 1 (CR1, CD35). Antibodies to these Knops system antigens are not uncommon. They are not haemolytic and do not reduce the survival of transfused incompatible red cells, but they are a nuisance in transfusion laboratories as they can cause an incompatible crossmatch and must be identified before they can be dismissed as clinically insignificant. Human red cell alloantibodies can be shown to be Knops system antibodies by the monoclonal-antibody-specific immobilization of erythrocyte antigens (MAIEA) test, using murine monoclonal anti-CR1. In addition to confirming that Kna, McCa, Sla and Yka are located on CR1, the MAIEA test was used to confirm that Csa is not on CR1. Red cells of the Helgeson phenotype, the null phenotype of the Knops system by conventional serological methods, have levels of Kna, McCa, Sla and Yka intermediate between those of alpha-chymotrypsin-treated cells (which lack Knops system antigens) and those of positive control cells. Level of expression of Knops system antigens is very variable and intensity of staining of immunoblots probed with monoclonal anti-CR1 correlated with strength of Knops system antigens, as determined by the MAIEA test. In individuals heterozygous for alleles producing different allotypes, separate bands representing each allotype on an immunoblot showed identical intensity of staining, suggesting that the quantity of CR1 on red cells is controlled, at least in part, by a locus independent of CR1. An analysis of CR1 on red cells of individuals who have made Knops system antibodies suggested that the Knops system antigens and the antibodies that detect them are complex and heterogeneous.

A new low-incidence antigen in the Kell blood group system: VLAN (KEL25).

A multilaboratory investigation has identified a new low-incidence antigen "VLAN' on the red cells of a blood donor. The VLAN antigen is destroyed by 2-aminoethylisothiouronium bromide treatment of the donor's red cells suggesting an association with the Kell system. Monoclonal antibody-specific immobilization of erythrocyte antigen analysis with anti-VLAN and with several mouse monoclonal antibodies directed at epitopes on the Kell glycoprotein gave positive results, indicating that the VLAN antigen is located on the Kell glycoprotein. The VLAN red blood cells have the common Kell phenotype: KEL:-1,2,-3,4,5,-6,7,-10,11,12,13,14,-17,18,19,-21,22,-23,-24. Additional serologic data indicate that the VLAN antigen is not part of any other ISBT blood group system, collection or series. A family study showed that the VLAN antigen is inherited since the red cells of two sisters and one niece of the propositus are also VLAN+. The ISBT Working Party on Terminology for Red Cell Surface Antigens has assigned VLAN to the Kell blood group system as KEL25 (number for computer listings 006025).

Investigation of the biochemical relationship between the blood group antigens Xga and CD99 (12E7 antigen) on red cells.

The biochemical relationship between the red cell antigens Xga and the MIC2 gene product, CD99--previously designated the 12E7 antigen--has been examined by immunoblotting and immunoprecipitation analyses of the protein molecules bearing these antigens. Immunoblotting of membrane components and Xga-immunoprecipitates with anti-Xga has shown that Xga antigen is carried on a broad band of apparent molecular weight (Mr)) 24,500-29,500, which consists of a dark stained component at M(r)24,500 and a more diffusely stained component at approximately M(r) 26,500-29,500. Immunoblotting of membrane components and 12E7-immunoprecipitates with 12E7, and RFB-1 and NaM123 which also recognise CD99, distinguished two bands of M(r) 30,000 and 32,000. A non-radioactive immunoprecipitation technique was employed, which uses chemiluminescence detection of biotin-labelled red cell proteins. The protein of M(r) 32,000, which carries CD99, was identified by this method and the red cell quantitative polymorphism of CD99 was demonstrated. When the Xga protein was precipitated from biotin-labelled red cells, a protein of M(r) 32,000 was coprecipitated. This suggests that the proteins carrying the Xga antigen and CD99 are associated in the membrane.

PBDX is the XG blood group gene.

We have identified the Xga antigen, encoded by the XG blood group gene, by employing rabbit polyclonal and mouse monoclonal antibodies raised against a peptide derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX. In indirect haemagglutination assays, these anti-peptide antibodies react with Xg(a+) but not Xg(a-) erythrocytes. In antibody-specific immobilization of antigen (ASIA) and immunoblot assays, the anti-peptide antibodies react with the same molecule as does human anti-Xga. Therefore, by its identity with PBDX, Xga is identified as a cell-surface protein that is 48% homologous to CD99 (previously designated the 12E7 antigen), the product of MIC2 which is tightly linked to XG. PBDX is renamed here XG.

Demonstration by the monoclonal antibody-specific immobilization of erythrocyte antigens assay that a new red cell antigen belongs to the Kell blood group system.

BACKGROUND: The Kell blood group system comprises 21 antigens residing on a red cell membrane glycoprotein of apparent M(r) 93,000. STUDY DESIGN AND METHODS: Serologic techniques were used to identify a new red cell antigen. The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay was used to identify the red cell membrane component carrying that antigen. RESULTS: A new high-frequency red cell antigen was identified and provisionally named RAZ. RAZ is absent from K.o red cells and from red cells treated with 2-amino-ethylisothiouronium bromide and is expressed weakly on McLeod phenotype cells. It differs from all other Kell system antigens, and no depression of other Kell system antigens on RAZ+ red cells was noticed. The RAZ antigen was shown by the MAIEA assay to be located on the Kell glycoprotein. CONCLUSION: RAZ is a new high-frequency antigen located on the Kell glycoprotein. The MAIEA assay is a very effective method of demonstrating the membrane structure carrying a red cell antigen.

Application of the MAIEA assay to the Kell blood group system.

The monoclonal antibody-specific immobilisation of erythrocyte antigens (MAIEA) test has been employed to investigate the Kell system using five monoclonal antibodies which recognise high frequency epitopes on the 93,000-molecular weight Kell glycoprotein: BRIC 18, BRIC 68, BRIC 107, BRIC 203 and 6-22. BRIC 107, which has anti-k-like (KEL2) specificity, identifies a distinct epitope, whilst competitive binding assays suggested that BRIC 203 (anti-Kpbc), BRIC 18, BRIC 68 and 6-22 (anti K14) comprise an overlapping set of epitopes. The MAIEA assay has been very successful in confirming the assignment of most of the Kell and para-Kell antigens to the Kell protein. Due to the competitive nature of the assay and the fact that the monoclonal antibodies bind to different regions, the results also suggest the relative positions of some of the Kell antigens on the Kell protein; these appear to be located in at least five spatially distinct regions.

Monoclonal antibody-specific immobilisation of erythrocyte antigens (MAIEA). A new technique to selectively determine antigenic sites on red cell membranes.

A new assay, the monoclonal antibody-immobilization of erythrocyte antigens (MAIEA) is described for the assignment of red cell antigens recognised by human allo-antisera, to particular membrane components of the red cell membrane. This technique detects tri-molecular complexes formed by the reaction of a human antibody and a mouse antibody with a particular red cell protein. A positive reaction, in an ELISA-type detection procedure, occurs if the epitopes recognised by the human and mouse antibodies are present on the same membrane component but different regions. The MAIEA assay has been developed to investigate blood group antigens and in this report its application to the study of the Lutheran, Yt and Kell blood group systems is described. The technique has been used to show that the Lu(a) and Lu3 antigens are carried on the Lutheran glycoprotein, Yta and Ytb on the enzyme acetylcholinesterase and K, k, Kpa and Kpb on the Kell glycoprotein.

Use of the MAIEA technique to confirm the relationship between the Cromer antigens and decay-accelerating factor and to assign provisionally antigens to the short-consensus repeats.

The MAIEA (monoclonal-antibody-specific immobilisation of erythrocyte antigens) assay has recently been developed for the assignment of red cell antigens, recognised by human alloantisera, to particular membrane components of the red cell membrane. This technique detects trimolecular complexes formed by the reaction of a human antibody and a mouse antibody with a particular red cell protein. A positive reaction, in an ELISA-type detection procedure, occurs if the epitopes to the human and mouse antibodies are present on the same membrane component but at different regions. In this report, we show how the MAIEA assay can be used to confirm the relationship between Cromer system antigens and the complement-regulatory protein, decay-accelerating factor (DAF, CD 55). In addition, the location of the antigens along the protein is postulated by using three anti-DAF monoclonal antibodies with specificities to different regions of DAF. Tca and Esa are assigned provisionally to the first short-consensus repeat (SCR), UMC to the second SCR, Dra to the third SCR and Cra, WESa and WESb to the fourth SCR or to the serine/threonine rich region of the DAF protein.

Release of Choline Metabolites from Human Platelets: Evidence for Activation of Phospholipase D and of Phosphatidylcholine-specific Phospholipase C.

In aspirin-treated platelets labelled by preincubation with [(3)H]-choline, enhanced release of both [(3)H]-choline and [(3)H]-choline phosphate resulted from stimulation by collagen or thrombin. No such release accompanied stimulation by ADP, platelet-activating factor or adrenaline. Release of [(3)H]-choline phosphate was entirely dependent on aggregate formation whereas release of [(3)H]-choline was reduced but not eliminated, if aggregation was prevented. The properties of [(3)H]-choline and [(3)H]-choline phosphate release indicated that both collagen and thrombin induced activation of phospholipase D in the absence of aggregate formation. Such activation was augmented if aggregate formation occurred. Aggregation induced by these two agonists also caused activation of phosphatidylcholine-specific phospholipase C. These effects were prevented in the presence of staurosporine and could also be induced by addition of a synthetic 1,2-diacylglycerol indicating a role for protein kinase C.

Factors affecting the observation of a synergistic response in platelet aggregation induced by pairs of excitatory agonists.

When aggregation is measured as the disappearance of single platelets synergistic interaction between excitatory agonist pairs can be observed using washed platelets in a modified Tyrode's medium or platelet-rich plasma anticoagulated with hirudin; but not using citrated platelet-rich plasma. For aggregation induced by the ADP/adrenaline agonist pair, both the observation of synergistic interaction and the sensitivity of the platelets to these agonists, is a function of extracellular [Ca(2+)]. Synergistic interaction and reduced sensitivity to the individual agonists, especially adrenaline, is observed when extracellular [Ca(2+)] > 100 µM. The data suggest that lower affinity binding of Ca(2+) to the glycoprotein IIb/IIIa complex may modulate platelet sensitivity to these excitatory agonists. The conditions used to resuspend the platelets also influences the nature of the response to the ADP/adrenaline agonist pair and the sensitivity of the platelets to these agonists. A synergistic response and/or reduced sensitivity to ADP is observed on resuspension in modified Tyrode's medium but does not occur on resuspension in citrated plasma or in plasma anticoagulated with hirudin. The factor responsible for enhancing sensitivity, and hence abolishing the synergistic response, is a species of low molecular weight (M(r) less than 25 KDa). It is neither citrate nor Ca(2+).

The use of high dose rate endobronchial brachytherapy to palliate symptomatic endobronchial recurrence of previously irradiated bronchogenic carcinoma.

Thirty-eight patients were treated with high dose rate endobronchial brachytherapy to palliate symptoms (cough, hemoptysis, fever, and/or shortness of breath) caused by endobronchial of previously irradiated (greater than or equal to 5000 cGy) bronchogenic carcinoma. The dose per fraction was 600 cGy at a radius of 1 cm from the center of the linear path of the source, and each patient received three fraction, each fraction separated by a 1-week interval. Twenty-nine patients (76%) had symptomatic improvement, 16 with complete and 13 with partial relief of symptoms. The likelihood of symptom relief was greater in those patients who had extra-bronchial tumor measuring less than 5 cm (15/15) compared to those with extra-bronchial tumor measuring greater than or equal to 5 cm (2/8). The median duration of symptom relief was 7.5 months. Repeat bronchoscopy done 3 months after brachytherapy revealed that 41% (11/27) had complete tumor regression and another 41% (11/27) had partial regression. Nine of 14 patients with post-obstructive atelectasis/pneumonitis had radiographic improvement. Twelve patients (32%) died from massive hemoptysis occurring 2-56 weeks (median 10 weeks) after brachytherapy. Location of the recurrence was the most important predictor of pulmonary hemorrhage. It occurred only in patients with recurrence in the right upper lobe, right mainstem, or left upper lobe bronchus. Whether this high rate of fatal pulmonary hemorrhage was a real phenomenon or a statistical fluke of small numbers remains an unanswered question.