Impaired release of a T-cell specific suppressor factor in rheumatoid arthritis.

Peripheral blood T cells from patients with rheumatoid arthritis (RA) and scleroderma (PSS) were assessed for their ability to release T-cell-specific suppressor activity (TRSA) upon incubation with a suppressor activating factor (SAF) derived from a human lymphoblastoid cell line (CEM). T cells from 11/20 (55%) RA patients exhibited impaired TRSA release in contrast to 1/12 (8%) of PSS patients. RA patients demonstrating impaired TRSA release exhibited more active arthritis than patients demonstrating normal TRSA release.     

Comparison of different PCR approaches for characterization of Burkholderia (Pseudomonas) cepacia isolates.

In this study, we evaluated three PCR methods for epidemiological typing of Burkholderia (Pseudomonas) cepacia--PCR-ribotyping, arbitrarily primed PCR (AP-PCR) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)--and compared them with pulsed-field gel electrophoresis. The analysis was performed with 31 isolates of B. cepacia, comprising 23 epidemiologically unrelated isolates and 8 isolates collected from the same patient during two episodes of bacteremia. Pulsed-field gel electrophoresis, ERIC-PCR, and AP-PCR identified 23 distinct types among the 23 unrelated isolates, while PCR-ribotyping only identified 12 strain types, even after AluI digestion of the amplification products. Among the eight isolates collected from the same patient, all typing techniques revealed two clones of strains. The day-to-day reproducibilities of PCR-ribotyping and ERIC-PCR were good, while greater day-to-day variations were noted in the fingerprints obtained by AP-PCR. We conclude that all three PCR techniques are useful for rapid epidemiological typing of B. cepacia, but ERIC-PCR seems to be more reproducible and discriminative.     

Effects of acrivastine,azelastine, cetirizine and noberastine on rat peritoneal mast cells

Inflammation Research -     

Intracellular T lymphocyte cytokine profiles in the aqueous humour of patients with uveitis and correlation with clinical phenotype

This study aimed to investigate whether T cells in aqueous humour are different in different types of uveitis and correlate with clinical phenotype. Patients with clinically different types of uveitis, but all displaying active anterior uveitis, were phenotyped and samples of aqueous humour (AH) and peripheral blood (PB) collected. Cells from AH and PB were separated by centrifugation and by density gradient centrifugation (to obtain mononuclear cells PBMC), respectively. Cells were activated with PMA and ionomycin in the presence of Brefeldin A, stained for surface markers and intracellular cytokines, and analysed by flow cytometry. The cytokine profile was correlated with the clinical phenotype. Increased percentages of interleukin (IL)-10+-, but not interferon (IFN)-gamma+ T lymphocytes were found in AH compared with PB in patients with acute anterior uveitis (AAU), FHC or chronic panuveitis (PU). There was a trend towards elevated levels of IL-10+ T cells in AH from patients with FHC compared with AH from acute uveitis and panuveitis patients. Increased levels of IL-10+ T cells in AH compared with PB were also found in samples from patients with isolated uveitis, but not those with associated systemic disease. Levels of cytokine-positive T cells were not associated with the use of topical steroids or to the severity of the anterior uveitis. While type I cytokine-producing T lymphocytes are present in AH during AU, the presence of increased proportions of IL-10+ T lymphocytes in AH from patients with uveitis may be indicative of an anti-inflammatory mechanism that may influence the type and course of ocular inflammation in these patients.     

Internet teleconferencing method for telepathology consultations from lung and heart transplant patients

Current Internet-based teleconferencing techniques allow a referring pathologist to transmit real-time images from a microscope to a consultant, while maintaining a verbal conversation using Internet telephony. In our study, 50 randomly selected transbronchial biopsies from lung allograft recipients and 58 randomly selected endomyocardial biopsies from heart transplant patients were diagnosed by consultant pathologists using Internet-based teleconferencing methods. The referring pathologists acquired the real-time video images from the biopsies using a light microscope equipped with a phototube adapter and a video camera. The consultant pathologists viewed the processed images on a video monitor at 800 x 600 resolution, using a standard microcomputer equipped with Netmeeting software, and directed the referring pathologist to move the slide under the microscopy and/or change image magnification. The validity of telepathology diagnoses was assessed with kappa coefficients. Consultations were completed in 5 to 15 minutes per case. Sound transmission was unreliable, and in approximately 25% of consultations the referring pathologist needed to "call back" to reestablish verbal communication. In all but 2 transbronchial biopsies there was agreement between the original diagnosis and the diagnosis by telepathology (kappa = 0.92). In 48 of 58 endomyocardial biopsies there was concordance between the 2 diagnoses (kappa = 0.692). Only 3 out of 10 of these discrepancies were clinically significant (kappa = 0.897). Internet-based teleconferencing techniques provide effective and relatively inexpensive tools for real time telepathology consultations. The technology is probably best suited for the study of small specimens from patients that require rapid diagnosis by a consultant.     

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.     

Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues

Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.     

Possible involvement of receptors in the entry of Kunjin virus into Vero cells

Summary The results obtained from electron microscopy, adsorbed and internalised virus assays and immunofluorescence studies supported that the most likely mode of entry of Kunjin virus into Vero cells was by receptor-mediated endocytosis. This was deduced indirectly from the time sequence of events that occurred. Electron microscopy revealed that endocytosis of the virus through coated vesicles had occurred. The adsorbed and internalised virus assay and immunofluorescence studies showed that there were two factors being recycled during endocytosis: the receptor for the virus and clathrin, the protein found on coated pits and vesicles. The study showed that clathrin was recycled first, followed by the receptor.     

Anorectal continence following manual and mechanical anastomosis suture. Results of a controlled study of rectal surgery

In a controlled clinical trial-manual vs. stapler anastomosis in rectal surgery-it was found that both suture techniques per se made no difference in the function of anal continence. The anal pressures at rest and sphincter contraction remained unchanged. A linear reduction of functional reservoir of the "neorectum" could be shown, which depended on the level and healing of the anastomosis. An anastomosis level at 6 cm from anocutaneous line is important for functional reasons. Anastomoses above this level do not cause any consequences for anal continence. Anastomoses below this level result in a reduced functional reservoir for at least 6 months. Within this period a decrease in anal continence is possible, especially in cases of disturbed healing of the anastomosis.