Comparison of flagellum and sonicate antigens for serological diagnosis of Lyme borreliosis

A sonicate antigen and two concentrations of a purified flagellum antigen ofBorrelia burgdorferi were compared for serological diagnosis of Lyme borreliosis by an enzyme immunoassay (EIA). Generally, the higher concentration of flagellum antigen was found to be superior to the lower concentration, which was diluted eight times compared to the higher concentration. The diagnostic sensitivity for IgG antibody detection increased from 13 % in the sonicate EIA to 31 % in the best flagellum EIA assay (p=0.01) in sera from patients with erythema migrans (n=70), and from 34 % to 55 % (p=0.01) in sera from patients with neuroborreliosis (n=77). However, the sensitivity for IgG in sera from patients with acrodermatitis chronica atrophicans (n=20) was high in both assays: 90 % in the sonicate EIA compared to 95 % in the flagellum EIA. Regarding IgM, there was no significant difference between the sensitivity of the assays in sera from any of the patient groups. The sensitivity values for IgM and IgG in cerebrospinal fluid (CSF) from patients with neuroborreliosis were also without significant differences. Sera and CSF from patients with meningitis/encephalitis of non-Borrelia etiology (n=35), multiple sclerosis (n=9) or syphilis (n=24), served as controls. The flagellum EIA showed a significantly improved specificity for IgG in CSF from controls with syphilis (p<0.01). It is concluded that purifiedBorrelia burgdorferi flagellum antigen is superior to a sonicate antigen, especially for serodiagnosis of the early stages of Lyme borreliosis.     

Nicotinamide reduces high secretion of IFN-gamma in high-risk relatives even though it does not prevent type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease suggested to be of a T helper (Th)1-like origin. This study aimed to investigate the Th1-like and Th2-like profile in high-risk individuals during the prediabetic phase and the immunologic effect of treatment with nicotinamide. High-risk first-degree relatives of T1D patients participating in the European Nicotinamide Diabetes Intervention Trial (ENDIT) were treated with either nicotinamide or placebo. Peripheral blood mononuclear cells (PBMC) were obtained during the prediabetic phase and close to the onset of manifest T1D and from nondiabetic high-risk individuals. Using the sensitive enzyme-linked immunospot (ELISPOT) technique to distinguish Th1-like from Th2-like lymphocytes, secretion of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) was analyzed from PBMCs spontaneously and after in vitro stimulation with the diabetes-associated autoantigens, glutamic acid decarboxylase 65 (GAD65, protein and peptide, aa 247-279), recombinant tyrosine phosphatase (IA-2), and heat shock protein (HSP, aa 437-460). High-risk individuals showed high spontaneous as well as autoantigen-induced IFN-gamma secretion. Secretion of IFN-gamma and the IFN-gamma/IL-4 ratio, induced by autoantigens, decreased in individuals developing T1D (p < 0.05), whereas nondiabetic individuals showed an increased IL-4 response (p < 0.05). Thus, a Th1-dominated cytokine profile observed in high-risk individuals inclined toward a diagnosis of diabetes. Nicotinamide caused decreased spontaneous (p = 0.05) and in vitro autoantigen-induced IFN-gamma secretion (p < 0.05) and may play a role in immune regulation, even though it has not been shown to prevent T1D.     

Slow axonal transport of soluble proteins and calpain in retinal ganglion cells of aged rabbits.

The rate of slow axonal transport of soluble proteins in retinal ganglion cells of the rabbit decreased with approximately 25% in aged (6 years) compared to previous estimates in adult (2 years) animals. Immunobinding of calpain to microtiter plates coated with a monoclonal antibody to mu-calpain was used to isolate labelled axonally transported mu-calpain from the nerve extracts. It was found that the distribution of labelled mu-calpain in the retrobulbar optic pathway was similar to the distribution profile of the slowly migrating phase of soluble proteins.     

Development of a Cell Patterning Technique Using Poly(Ethylene Glycol) Disilane

A simple technique for controlling cell adhesion on glass substrates by surface modification using a commercially available poly(ethylene glycol) (PEG) disilane, which can bind directly to glass in a single step, in combination with photolithographic micropatterning, was developed, characterized, and evaluated for patterning of HepG2 hepatoma cells and 3T3 fibroblasts. The optimal concentration of PEG-disilane for surface modification was 5 mM, and patterning of strongly adherent cells such as HepG2 required the chelation of divalent metal cations in order to inhibit nonspecific binding and cell aggregation. Whereas the average thickness of the PEG-disilane layer was 18±3.5 nm, the perimeters of patterned areas of exposed glass exhibited ridges of average height 857±50 nm, which may have aided in constraining cell spreading and migration. Although unpatterned PEG-treated substrates were hydrophilic (contact angle 46±1°), micropatterned surfaces behaved as if they were somewhat hydrophobic (contact angle 90°), necessitating special protocols for preventing deleterious dewetting of cells. For optimized protocols, the probability of adhesion of HepG2 cells to a patterned area of exposed glass was almost 15 times higher than the probability of adhesion to a PEG-treated background region of equal area. Our technique is useful for short- to intermediate-term patterning of cell or tissue morphology, e.g., for investigation of the effects of cell–cell interactions or cell geometry.     

KIR down-regulation on NK cells is associated with down-regulation of activating receptors and NK cell inactivation

We previously reported that killer cell immunoglobulin-like receptors (KIR) could be down-regulated from the surface of T cells. Here, we show that KIR down-regulation is also induced on the surface of natural killer (NK) cells upon ligand binding. Common down-regulation characteristics are found on these two cell types: a slow kinetics and a phenomenon observed for long inhibitory forms only. Importantly, KIR down-regulation on NK cells is associated with a down-regulation of activating receptors (CD16, CD2 and 2B4) as well as with a lack of cell responsiveness (antibody-dependent and natural killing activities). This unresponsive state was not observed for MHC-restricted T cells. Our data implicate that, in addition to prevention of the immediate target cell lysis, KIR-MHC class I interactions may also regulate the subsequent NK cell cytotoxic activity. This observation opens new perspectives in the understanding of NK cell regulation.     

Die Wirkung von Angiotensin und Metopiron auf den Gehalt an Glucose-6-Phosphat-Dehydrogenase und Succinat-Dehydrogenase in der Zona glomerulosa der Nebennierenrinde intakter Ratten

Summary It was shown that angiotensin administered to the intact rat increases the activity of the glucose-6-phosphate and succinic acid dehydrogenases in the zona glomerulosa and that this effect is enhanced by methopyrapone. It would seem that the hexose-monophosphatase shunt is of importance for the steroidogenesis also in the zona glomerulosa. It is possible that the shunt is activated by angiotensin in a way similar to that by which it is stimulated by ACTH in the inner zones. The activation of the succinic acid dehydrogenase indicates an stimulation of the Krebs cycle by angiotensin. The enhancing effect of methopyrapone remains so far unexplained.

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Der AG Ciba danken wir für die Unterstützung bei diesen Untersuchungen.     

Evidence for recessive inheritance of myopia

Data are presented on the family distribution of nearsightedness, including the frequency of the disorder in the children of two myopes. The previous literature is reviewed, embracing several quantitative family or population studies in addition to extensive pedigree material. It is concluded that there is substantial evidence that recessive inheritance is the principal mechanism of transmission, and that most instances of myopia probably fall into that category.     

Evaluation of the E test for susceptibility testing of anaerobic bacteria.

The susceptibilities of 105 clinical isolates of anaerobic bacteria were determined by a new method, the E test (AB Biodisk, Solna, Sweden), and were compared with the MICs for these organisms obtained by the reference agar dilution method by using supplemented brucella and Wilkins-Chalgren agars. The E test is a plastic strip with a predefined antibiotic gradient immobilized on one side and a MIC interpretive scale printed on the other side. Strips with cefoxitin, cefotaxime, imipenem, penicillin, metronidazole, and clindamycin were used in this study. A suspension of the test strain equal to the visual turbidity of a no. 0.5 McFarland standard was prepared and swabbed onto a 150-mm-diameter plate. The strips were applied in a radial fashion, and the plates were incubated under anaerobic conditions. After growth had occurred, an ellipse of inhibition was seen around each strip. At the point of intersection of the ellipse with the strip, the MIC was read from the interpretive scale. For most antibiotic-organism combinations, the ellipse was clear and the endpoint was sharp. The E-test MICs were interpreted after overnight and 48-h incubation for 58 of the strains. After overnight incubation, 87% of the E-test MICs were within 1 dilution of the agar dilution MICs, and 98% were within 2 dilutions. After 48 h of incubation, agreement was 86 and 97% respectively. E-test MICs obtained for the Bacteroides fragilis group after overnight incubation were more comparable than those obtained after 48 h of incubation to agar dilution MICs determined at 48 h for all drugs except clindamycin. On brucella agar, there was a 2% categorical discrepancy rate between the E-test MICs and agar dilution MICs, which occurred mostly with cefoxitin. The E test is easy to perform and read, is suitable for all anaerobes, can be used to test single patient isolates as needed, and offers the laboratory a reliable method for susceptibility testing of anaerobic bacteria.     

A Double-Blind, Multicenter Immunotherapy Trial in Children, Using a Purified and Standardized Cladosporium herbarum Preparation II. In Vitro Results

This double-blind immunotherapy trial in children, using a purified and standardized Cladosporium herbarum allergen preparation, has shown that children with mould asthma and/or rhinoconjunctivitis, responded to immunotherapy with a decrease in specific IgE and a significant increase in specific IgG. There was a marked increase in the ratio specific IgG/specific IgE as a result of active treatment. IgE-CRIE radiostaining patterns showed no pronounced changes after 10 months' active treatment and no "new sensitivities" could be detected in the studied patients. IgG-CRIE radiostaining, primarily directed towards the important allergens, was significantly increased in the active group and particularly towards Ag-12 (partially identical to a previously described major allergen in Cladosporium herbarum, Ag-54). Children treated with histamine placebo showed no change in antibody patterns during 10 months of treatment.     

Enzyme-linked immunosorbent assay for immunological diagnosis of human tularemia.

The enzyme-linked immunosorbent assay (ELISA) was applied for immunological diagnosis of human tularemia, using lipopolysaccharide from Francisella tularensis as antigen. Sera collected from patients, healthy individuals, and vaccinated volunteers were investigated for antibodies against F. tularensis by ELISA and tube agglutination. In ELISA all sera were titrated with a polyspecific anti-immunoglobulin enzyme conjugate. A limited number of consecutive sera from individual patients were also investigated for immunoglobulin G (IgG) and IgM antibodies by means of immunoglobulin class-specific conjugates. On an average ELISA was more than 10-fold as sensitive as tube agglutination. Two weeks after onset of disease, sera from patients had significantly higher titers in ELISA than sera from healthy controls. High titers persisted after more than 2 years. Significant amounts of both IgG and IgM antibodies were present within 1 to 2 weeks after infection. The antibody activity increased during the first month, without any significant change of the relation between IgG and IgM titers. After 2.5 years the IgG/IgM titer ratio of sera from patients was significantly increased. Within 6 weeks after vaccination sera from about half of the vaccinees had significantly elevated titers in ELISA. Titers observed after vaccination were generally lower than those found after infection. An elevated ELISA titer can be of diagnostic importance by the end of the first week of illness. A significant increase of titer in consecutive serum samples indicates a diagnosis of tularemia. Determination of IgG and IgM antibodies may be of value in determing whether a positive titer of a single serum sample is of longstanding or recent origin.