Immune complexes in early arthritis. L Detection of immune complexes before rheumatoid arthritis is definite

Fifty-three patients with early arthritis were studied longitudinally for up to 3 years. During this time, 24 developed sufficient features for definite rheumatoid arthritis (RA) to be diagnosed. The other (arthralgia patients) differed from the RA patients as, in the majority, C-reactive protein and ESR were normal and anti-nuclear antibodies or rheumatoid factors were rarely found. Moreover, in time their signs and symptoms improved or disappeared. Circulating immune complexes were detected in both groups of patients by the platelet aggregation test whereas complexes detected by abnormal Clq-binding activity were found mainly in the RA patients. Platelet-aggregating complexes were usually present in the first samples studied and disappeared in the arthralgia patients with recovery from their symptoms. In the RA patients, Clq-binding complexes appeared simultaneously or later than platelet-aggregating complexes but both tests were positive several months before RA could be diagnosed. These results suggest that immune complexes are one of the first immunological abnormalities to appear in patients with arthritis. Although the constituent antigen and antibody of complexes detected by either test are unknown, their possible nature is discussed.     

Identification of antibody epitopes in the CB-11 peptide of bovine type II collagen recognized by sera from arthritis-susceptible and -resistant rhesus monkeys.

Sera from eight rhesus monkeys that had been immunized with native bovine type II collagen were tested for antibodies to cyanogen bromide peptides (CB peptides) of type II collagen by Western blotting. The monkeys produced IgG antibodies to a number of different CB peptides, with five out of eight animals producing antibodies to the CB-11 peptide (four arthritic, one non-arthritic). Antibody epitopes on the CB-11 peptide of bovine type II collagen recognized by these sera were investigated by epitope mapping. Peptides (8-mers overlapping by seven amino acids) representing the CB-11 region were synthesised and the sera screened for binding to these peptides to determine areas of high IgG antibody binding to this region of type II collagen. The profiles obtained were not identical, though there were some epitopes that were commonly recognized. Antibodies to one epitope, also present in human type II collagen, were found only in the sera of two animals with the severest arthritis. The technique of epitope mapping has successfully identified a number of epitopes within the CB-11 peptide of type II collagen recognized by antibodies from bovine type II collagen-immunized monkeys. Studies on the relevance of responses to the identified epitopes can now be undertaken.     

Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens

The chemical composition of the outer membrane fractions (OMFs) of Eikenella corrodens strains 23834 and 470 as well as the strain 23834 lipopolysaccharide (LPS) was determined. The OMFs were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMFs of strains 23834 and 470, free of cytoplasmic membrane components, were found to contain 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate, and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis both OMFs contained one major peptide determined to be 33,500 daltons for the strain 23834 OMF, and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic, and lesser amounts of octadecanoic acids. Transmission electron microscopic examination of the OMFs revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was found to be composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose could be detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed the E. corrodens LPS to resemble other smooth-type LPSs. Transmission electron microscopic examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a "classical," i.e., enteric, type of LPS.     

Cigarette smoke and phagocyte function: effect of chronic exposure in vivo and acute exposure in vitro.

Phagocytic function was studied in mice chronically exposed to cigarette smoke, and the effects of in vitro exposure to cigarette smoke on macrophage activity were also assessed. Cultures of radiolabeled Pseudomonas aeruginosa were employed to investigate phagocyte activity in vivo and in vitro. Mice were exposed on weekdays to fresh cigarette smoke for periods up to 37 weeks and the bactericidal and clearance activity of their lungs was measured. Both pulmonary clearance and bactericidal activity was impaired. The clearance of intravenously injected bacteria from the blood of smoke-exposed mice occurred at the same rate as in control mice, but the accumulation of radiolabel by the liver was decreased. In addition, the rate of elimination of radiolabel from the liver was less than the controls. Macrophages exposed to cigarette smoke in vitro initially had a depressed phagocytic rate, but if phagocytosis over a prolonged period was measured it was eventually enhanced over the rate of control macrophages. The vapor phase of cigarette smoke could also transiently inhibit and then enhance the phagocytic activity.     

Migration of human lymphocytes. I. A model using the mouse as host.

The distribution of radioactivity after the intravenous injection of 51Cr-labelled human lymphocytes has been examined in normal mice, irradiated mice, mice treated with anti-platelet antiserum and in mice treated with colloidal carbon. Pre-treatment with carbon and anti-platelet antiserum appears to protect the human lymphocytes from uptake by the host's reticuloendothelial system (RES). Comparison of tissue radioactivity in carbon-treated mice after the injection of viable human lymphocytes with that found after the injection of dead cells and soluble or insoluble cell debris showed that radioactivity recovered in the spleen and lymph nodes is primarily due to the migration of viable lymphocytes into these tissues. Thus the measurement of radioactivity in lymph nodes of carbon-treated mice after the injection of 51Cr-labelled human lymphocytes can be used as a model of these lymphocytes' ability to migrate into the lymph nodes during recirculation and to study factors influencing this migration.     

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

House dust mite allergen avoidance and self-management in allergic patients with asthma: randomised controlled trial

BACKGROUND: The efficacy of bed covers that are impermeable to house dust mites has been disputed. AIM: The aim of the present study was to investigate whether the combination of 'house dust mite impermeable' covers and a self-management plan, based on peak flow values and symptoms, leads to reduced use of inhaled corticosteroids (ICS) than self-management alone. DESIGN OF STUDY: Prospective, randomised, double blind, placebo-controlled trial. SETTING: Primary care in a south-eastern region of the Netherlands. METHOD: Asthma patients aged between 16 and 60 years with a house dust mite allergy requiring ICS were randomised to intervention and placebo groups. They were trained to use a self-management plan based on peak flow and symptoms. After a 3-month training period, the intervention commenced using house dust mite impermeable and placebo bed covers. The follow-up period was 2 years. Primary outcome was the use of ICS; secondary outcomes were peak expiratory flow parameters, asthma control, and symptoms. RESULTS: One hundred and twenty-six patients started the intervention with house dust mite impermeable or placebo bed covers. After 1 and 2 years, significant differences in allergen exposure were found between the intervention and control groups (P<0.001). No significant difference between the intervention and control groups was found in the dose of ICS (P = 0.08), morning peak flow (P = 0.52), peak flow variability (P = 0.36), dyspnoea (P = 0.46), wheezing (P = 0.77), or coughing (P = 0.41). There was no difference in asthma control between the intervention and control groups. CONCLUSION: House dust mite impermeable bed covers combined with self-management do not lead to reduced use of ICS compared with self-management alone.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Oocyte morphology predicts outcome of intracytoplasmic sperm injection

To examine the influence of cytoplasmic morphology on the success rate of intracytoplasmic sperm injection (ICSI), the morphology of 837 metaphase II oocytes was assessed after cumulus stripping. The main abnormalities detected were excessive granularity, cytoplasmic inclusions such as vacuoles, smooth endoplasmic reticulum clustering and refractile bodies. Microinjection was performed in 538 oocytes with normal cytoplasm, 142 out of 161 with excessive granularity and 112 out of 138 with cytoplasmic inclusions. Very poor oocytes were not injected. No difference was found in fertilization rate. The embryos achieved cleaved normally and a similar number of good quality embryos among the three groups was noted. The outcome of transfer of embryos derived solely from normal oocytes (group A: 72 patients, 183 embryos) was compared with those from oocytes with cytoplasmic abnormalities (group B: 34 patients, 85 embryos). In group A, 17 clinical pregnancies (24% per patient, implantation rate 10%) were established. In group B, only one clinical pregnancy (3% per patient, implantation rate 1%) was established, from the transfer of embryos derived from oocytes with homogeneous granularity of the cytoplasm. No pregnancy resulted following the transfer of embryos from eggs with cytoplasmic inclusions. The difference was statistically significant. The outcome of ICSI is dependent on the quality of the oocytes retrieved. Normal fertilization and early embryo development were achieved in oocytes with abnormal cytoplasm morphology, but the resulting embryos failed to demonstrate the same implantation potential as those derived from oocytes with normal cytoplasm.