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.     

Tyrosine phosphatase-like protein (IA-2) and glutamic acid decarboxylase (GAD65) autoantibodies: a study of Chinese patients with diabetes mellitus

Type 1 diabetes in most Asian populations may not have a salient autoimmune basis when assessed with single determinations of the major markers, islet cell antibodies (ICAs) and glutamic acid decarboxylase antibodies (GAD65ab). With the inclusion of antibodies to tyrosine phosphatase-like protein IA-2 (IA-2ab) as an additional major marker, we re-examined autoimmune diabetes in a group of Chinese patients. We studied 272 subjects at various stages of disease with blood samples procured for biochemical analysis. ICAs were measured by immunofluorescence, GAD65ab and IA-2ab by radioimmunoassay. Sixty-seven patients fulfilled clinical diagnosis of type 1 diabetes and the remaining 205 patients were type 2. Prevalence of single autoantibody type in recent-onset type 1 diabetes ( < 1 year duration; n = 47) showed 10.6% with ICAs, 44.7% GAD65ab and 36.2% IA-2ab. GAD65ab account for more than two-thirds of the markers found in type 1 diabetes. Combined analysis further showed that 51.1% had at least one antibody type, 31.9% with two or more antibodies and 8.5% with all three antibodies. Islet autoimmunity presence in childhood-onset type 1 diabetes improved with the addition of IA-2ab, though less impact was seen in the adult-onset. Similarly, combined analysis for type 2 patients with recent diabetes showed a modest increase to 13% with islet autoimmunity compared to 8% when assessed by GAD65ab alone. Combining IA-2ab and GAD65ab assays results detected slightly more immune-mediated diabetes, compared to using a single GAD65ab determination. Non-autoimmune causes need to be considered in the pathogenesis of type 1 diabetes in Chinese, particularly in adults.     

Hepatitis C virus (HCV) genotypes in the Caribbean island of Martinique: evidence for a large radiation of HCV-2 and for a recent introduction from Europe of HCV-4

Molecular epidemiological studies of hepatitis C virus (HCV) in the Caribbean may help to specify the origin and spread of HCV infection. Indeed, the Caribbean population is intermixed from European and African origins and geographically close to the American continent. We characterized HCV genotypes in the Caribbean island of Martinique. HCV genotypes were analyzed by sequencing or reverse hybridization in the 5' noncoding region (5'NC) in 250 HCV-monoinfected and 85 HCV-human immunodeficiency virus (HIV)-coinfected patients. In addition, sequencing in the nonstructural 5B (NS5B) gene was required to determine the subtype or to perform phylogenetic analysis in selected samples. Genotypes 1 to 6 were found, respectively, in 84.4, 6.8, 5.2, 2.8, 0.4, and 0.4% of 250 HCV-monoinfected patients and in 71.7, 7.1, 15.3, 5.9, 0, and 0% of 85 HCV-HIV-coinfected patients. HCV-1b was found in 66.4% of the HCV-monoinfected patients and was associated with blood transfusion, whereas HCV-1a was detected in 41.2% of the HCV-HIV-coinfected patients and was associated with intravenous drug use (IVDU). The HCV-3 strains belonged to subtype 3a and were linked to IVDU. Phylogenetic analyses were focused on HCV-2 and HCV-4, which are common in Africa. Two opposite patterns were evidenced. NS5B sequences from 19 HCV-2 isolates were affiliated with many different subtypes described either in Europe or in West Africa, suggesting an ancient radiation. In contrast, seven of the nine HCV-4 NS5B sequences ranged within HCV-4a and HCV-4d clusters spreading in continental France by the IVDU route. Epidemiological data demonstrate the recent introduction of HCV-4a and -4d subtypes into the Caribbean.     

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.     

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.     

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.     

Contribution of magnetic resonance microscopy in the 12-week neurotoxicity evaluation of carbonyl sulfide in Fischer 344 rats

In this carbonyl sulfide (COS) study, magnetic resonance microscopy (MRM) and detailed light microscopic evaluation effectively functioned in parallel to assure that the distribution and degree of pathology in the brain was accurately represented. MRM is a powerful imaging modality that allows for excellent identification of neuroanatomical structures coupled with the ability to acquire 200 or more cross-sectional images of the brain, and the ability to display them in multiple planes. F344 rats were exposed to 200-600 ppm COS for up to 12 weeks. Prior to MRM, rats were anesthetized and cardiac perfused with McDowell Trump's fixative containing a gadolinium MR contrast medium. Fixed specimens were scanned at the Duke Center for In Vivo Microscopy on a 9.4 Tesla magnetic resonance system adapted explicitly for microscopic imaging. An advantage of MRM in this study was the ability to identify lesions in rats that appeared clinically normal prior to sacrifice and the opportunity to identify lesions in areas of the brain which would not be included in conventional studies. Other advantages include the ability to examine the brain in multiple planes (transverse, dorsal, sagittal) and obtain and save the MRM images in a digital format that allows for postexperimental data processing and manipulation. MRM images were correlated with neuroanatomical and neuropathological findings. All suspected MRM images were compared to corresponding H&E slides. An important aspect of this study was that MRM was critical in defining our strategy for sectioning the brain, and for designing mechanistic studies (cytochrome oxidase evaluations) and functional assessments (electrophysiology studies) on specifically targeted anatomical sites following COS exposure.     

Study of discharge communications from hospital doctors to an inner London general practice

In inner London patients are now being discharged from hospital earlier to be cared for by the community services. In this study the general practitioners in one inner London practice were asked to evaluate discharge communications from hospital doctors. The general practitioners were dissatisfied with the delay in receiving over one third of the letters and with the content of almost a fifth. They also felt that delay and lack of detail affected their management in 24% of cases. They would have liked more information in the letters, particularly about drug regimens. Some suggestions for improvement in written discharge communications are made.     

Comparison of Two Culture Methods for Detection of Tobramycin-Resistant Gram-Negative Organisms in the Sputum of Patients with Cystic Fibrosis

A culture method utilizing quantitative plating on antibiotic-containing media has been proposed as a technique for the detection of tobramycin-resistant organisms that is more sensitive than standard methods. Typical sputum culture methods quantitate the relative amounts of each distinct morphotype, followed by antibiotic susceptibility testing of a single colony of each morphotype. Sputum specimens from 240 cystic fibrosis patients were homogenized, serially diluted, and processed in parallel by the standard method (MacConkey agar and OF basal medium with agar, polymyxin, bacitracin, and lactose) and by plating on antibiotic-containing media (MacConkey agar with tobramycin added at 25 microg/ml [MAC-25] and 100 microg/ml [MAC-100]). MICs of tobramycin were determined for all Pseudomonas aeruginosa isolates by broth microdilution. Growth of P. aeruginosa on MAC-25 was considered to be equivalent to a tobramycin MIC of > or = 16 microg/ml, and growth on MAC-100 was considered to be equivalent to a tobramycin MIC of > or = 128 microg/ml. Analysis of method-specific detection rates showed that tobramycin-containing medium was more sensitive than the standard method for the detection of tobramycin-resistant P. aeruginosa, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans but was less sensitive for the detection of Burkholderia cepacia than the standard method. When MICs for P. aeruginosa that grew on tobramycin-containing medium were tested by broth microdilution, the MICs for 28 of 121 strains (23%) growing on MAC-25 and 22 of 56 strains (39%) growing on MAC-100 were MICs < 16 and < 128 microg/ml, respectively. Addition of a tobramycin-containing MacConkey plate to the routine media for sputum culture may provide additional, clinically relevant microbiologic data.