Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies

Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA.     

神经元性一氧化氮合酶活性在app/ps1双转基因AD模型小鼠皮质和海马的分布

本实验用 4～ 5月龄和 15～ 16月龄的 app/ ps1dtg小鼠各 5只 ,同龄野生型小鼠每个年龄组各 5只 ,用β-NADPH组织化学染色显示神经元一氧化氮合酶活性 ,刚果红组织学染色显示神经炎性斑 ,无偏性体视学计量皮质和海马神经炎斑的总体积 ,研究神经元一氧化氮合酶活性在 app/ ps1双转基因 (app/ ps1dtg) AD模型小鼠大脑皮质和海马的异常分布 ,探讨其在该模型小鼠及 AD患者脑内病理改变中的作用。结果显示 ,n NOS阳性神经元在各组小鼠皮质和海马内的分布没有区别 ,4～ 5月龄 app/ps1dtg小鼠皮质和海马可见神经炎斑 (NP)和营养不良性 NOS阳性神经突 (DTN) ;15～ 16月龄 app/ ps1dtg小鼠皮质和海马内NP的体积分别是 4～ 5月龄 app/ ps1dtg小鼠皮质 NP的 5倍 (P<0 .0 0 5 )和 8倍 (P<0 .0 0 1) ;15～ 16月龄 app/ ps1dtg小鼠皮质内 DTN的体积明显增大 (P<0 .0 1) ;且伴有 NOS阳性神经元形态的改变及海马 CA1 区 NOS阳性神经元数量的减少 (P<0 .0 5 ) ,DTN的形成与 NP呈正相关关系 (r=0 .85 ,P<0 .0 5 )。本实验结果表明 ,app/ ps1dtg小鼠能够模拟 AD患者脑内 NOS阳性神经元的病理改变 ,DTN的形成可能与 Aβ的毒性作用有关 ,DTN产生的 NO可能参与 app/ ps1dtg小鼠和 AD的神经病理过程。     

Computer image sperm selection as a novel approach to subzonal insemination in the human

Utilizing real-time computer image analysis, individual spermatozoawere selected using microaspiration. Selection criteria werebased on potential hyperactivation motility characteristics;the amplitude of lateral head displacement >7.5 µm,curvilinear velocity >70 µm/s and linearity of <30%.For this pilot study, 16 patients (eight in each group) wererecruited. Using subzonal insemination (SUZI), up to five (mean= 4.4 ± 0.3) spermatozoa selected using computer-imagesperm selection (CISS) were microinjected, or up to 15 (mean= 12.8 ± 1.3 SD) unselected spermatozoa. In the groupwhich utilized CISS, 28 out of 49 (57%) oocytes were fertilizedcompared with 13 out of 52 (25%) utilizing conventional SUZI(P < 0.04); polyspermy was 20% (n = 10) and 2% (n = 1) respectively.CISS with SUZI showed increased efficiency in achieving fertilizationand is a novel approach to studying individual sperm functionin a sperm egg bioassay where gamete ratios are close to unity.     

Inhibitory monoclonal antibody against a (myristylated) small-molecular-weight antigen from Plasmodium falciparum associated with the parasitophorous vacuole membrane.

A small-molecular-weight antigen that occurs in asexual blood stages in synchronized cultures of Plasmodium falciparum was detected by a monoclonal antibody which inhibits parasite growth in vitro. This antigen, QF116, showed a molecular weight of 15,000 in parasite strain FCR-3K+ from The Gambia and 19,000 in strain FCQ-27 from Papua New Guinea. The protein did not show significant glycosylation by galactose or glucosamine labeling but was found to be acylated by myristic acid. By using immunogold labeling and electron microscopy, the location of the antigen could be attributed to the parasitophorous vacuole membrane and to inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell.     

Ocular inflammatory effects of intravitreally injected tumor necrosis factor-alpha and endotoxin

Intravitreal injection of human recombinant tumor necrosis factor-alpha (TNF) induced inflammation in the rabbit eye characterized by dilation of blood vessels in the iris, disruption of the blood-ocular barriers, infiltration of inflammatory cells into the anterior chamber, and accumulation of prostaglandin E in intraocular fluids. Inflammation first appeared on day 1, increased on day 2, and remained elevated on day 7. The inflammatory eell infiltrate in the anterior segment of the eye was largely monocytic on days 1 and 2; by day 7 large numbers of lymphocytes were also present. TNF-induced ocular inflammation therefore differed from that reported for intravitreally injected endotoxin in terms of time course and the types of inflammatory cells in the aqueous humor. In a series of experiments in which combinations of TNF and endotoxin were used, intravitreal injection of TNF, 24 h after a low dose ofEscherichia coli endotoxin, produced no more inflammation than that produced by TNF following an injection of endotoxin vehicle. However, if TNF was injected 24 h before endotoxin, the resulting inflammation was greater than that observed in animals given TNF followed by endotoxin vehicle.     

Correlates of distress in children at risk for affective disorder: exploring predictors in the offspring of depressed and nondepressed mothers

BACKGROUND: Efforts to understand the correlates of psychological distress in children frequently examine possible correlates in samples of children who are selected for high levels of distress. The propose of this study was to compare distress correlates in a sample with depressed mothers, and thus at high-risk for distress, to a low-risk sample. METHODS: Examining data from part of a larger project, the association of children's depressive symptoms and internalizing and externalizing problems to maternal depression level, life stress, verbal ability, and the experience of a traumatic event were examined in a series of regression equations. RESULTS: Results indicated that children's depressive symptoms, rather than internalizing and externalizing problems, tended to be most consistently related to maternal variables, and also suggested that any experience of maternal depressive symptoms was associated with child problems. It was also found that child depressive symptoms were correlated with life events, but only for nondepressed mothers, and that at-risk children with higher levels of verbal ability were significantly less likely to report experiencing depressive symptoms and internalizing problems than were those with lower levels of verbal ability. LIMITATIONS: Because these data are preliminary, further research examining a broader array of variables is important. CONCLUSIONS: These results suggest the need for different models of these processes in different populations of children.     

Enhancing a primary care environment: a case study of effects on patients and staff in a single general practice

BACKGROUND: Few studies have examined the effect on patients and staff of the physical environment in primary care facilities. AIM: To explore changes in patient and staff satisfaction, patient anxiety, and patient-doctor communication when a GP surgery moves from old premises to enhanced purpose-built accommodation. DESIGN OF STUDY: Questionnaire surveys, interviews, and focus groups pre- and post move. SETTING: An urban general practice in Bristol. METHOD: Patient questionnaires assessed anxiety (Spielberger State-Trait Anxiety Inventory; STAI), satisfaction with the environment, and communication during the consultation. Staff questionnaires assessed satisfaction with the environment and job satisfaction. Qualitative methods explored patient and staff views in more depth. RESULTS: A total of 1118 pre-move and 954 post-move patient questionnaires showed significant increases in satisfaction scores for reception/waiting areas (mean 6.46, 95% confidence interval [CI]=5.97 to 6.95) and consulting rooms (mean 3.80, 95% CI=3.44 to 4.15) in the new premises. Patients' satisfaction with patient-doctor communication also increased (mean 0.88, 95% CI=0.30 to 1.46) and anxiety scores were significantly reduced before and after the consultation in the new premises compared with the old (STAI mean difference before consultation 0.72, 95% CI=0.37 to 1.08; mean after consultation 0.37, 95% CI=0.03 to 0.72). Patients highlighted the increased space and light, more modern appearance, greater comfort, and novel works of art in the new surgery. Staff workplace satisfaction increased significantly after moving and remained higher than in the old building. CONCLUSION: This large-scale study examining the effects of a UK primary care environment on patients and staff shows that an enhanced environment is associated with improvements in patients' perception of patient-doctor communication, reduction in anxiety, and increases in patient and staff satisfaction.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

Bactericidal activity of rabbit serum containing immunoconglutinin

When lymphocytes from hypersensitive animals are incubated with antigen, biologically active substances are formed which inhibit the migration of mesenchymal cells from normal animals.

These substances were tested by intradermal injection in guinea-pigs and rabbits. The supernatants from incubation of lymphocytes with a high dose of antigen caused immediate pallor which lasted several hours. Later there was a macroscopic inflammation maximal at 24 hours. The histology was characteristic of a delayed hypersensitivity reaction.

The injection of the supernatant from hypersensitive lymphocytes incubated with a small dose of antigen caused little or no pallor and was not followed by a delayed inflammatory reaction. Injection of this supernatant together with the antigen did not potentiate or alter the reaction, in contrast to in vitro experiments where the inhibition of the migration by this supernatant was potentiated by antigen.

Besides this factor a distinct factor occurs in extracts and supernatant fluids of lymphocytes cultivated without antigen and those from control animals, which increases vascular permeability. This substance is probably identical with the lymph node permeability factor (LNPF). The possible role of these biologically active substances in the mechanism of delayed type hypersensitivity is discussed.

     

The influence of deep body temperatures and skin temperatures on respiratory frequency in the pig

1. The influences on respiratory frequency of ambient temperature, the temperature of the skin, the temperature and humidity of the inspired air, hypothalamic temperature, the temperature of the spinal cord, rectal temperature and some temperatures in the abdomen have been studied in the pig.2. At a constant ambient temperature the effect on respiratory frequency of heating a thermode in the hypothalamus was modified by the temperature of the skin of the trunk which was varied independently by means of a temperature-controlled coat. A cold skin inhibited panting; a warm skin enhanced panting. The effect of heating a thermode over the spinal cord was similarly modified by skin temperatures.3. Simultaneous heating of thermodes in the hypothalamus and spinal cord increased respiratory frequency more than heating either alone, and in a warm environment the rectal temperature influenced the extent to which respiratory frequency increased on heating the thermodes.4. Cooling the thermodes decreased respiratory frequency in a warm environment and the cooling of one thermode enhanced the effect of cooling the other.5. At a constant trunk skin temperature the effect on respiratory frequency of heating the thermode in the hypothalamus depended on ambient temperature.6. Changing the temperature of thermodes in the abdomen did not affect respiration nor was there any evidence that the temperature and humidity of the inspired air had a direct effect on respiration.