[Mycoplasma pneumonia affecting the central nervous system]. / Mycoplasma pneumonia-infektion med affektion af centralnervesystemet.

The authors describe a case of Guillain-Barré syndrome and a case of encephalitis, both with serological proven Mycoplasma pneumoniae infection. In the patient with Guillain-Barré syndrome, a 49 year old male who had neurological sequelae five months after discharge, a throat swab was PCR positive for M pneumonia. The patient with encephalitis was discharged 19 days after admission without sequelae.

[Mycoplasma pneumoniae infection. Pulmonary and extrapulmonary manifestations]. / Mycoplasma pneumoniae infektion. Pulmonale og ekstrapulmonale manifestationer.

Mycoplasma pneumoniae is known to cause a mild disease of the upper respiratory tract and uncomplicated pneumonia in infected individuals. Serious pulmonary and extrapulmonary complications are reported with increasing frequency. In this paper pulmonary, neurological, haematological, dermatological, cardial, gastrointestinal and renal complications are reviewed. Since the diagnosis requires laboratory confirmation and indeed is often retrospective, knowledge of the less known pulmonary and systemic manifestations is important to make proper diagnostic decisions and to ensure proper medical treatment.

Prospective study of anticardiolipin antibodies in immunized and untreated women with recurrent spontaneous abortions.

OBJECTIVE: To investigate whether active leukocyte immunization increases levels of anticardiolipin antibodies in women with recurrent spontaneous abortions. To assess the impact of anticardiolipin antibodies on pregnancy outcome in these women. DESIGN: Patients who had received various treatments in an ongoing randomized trial were studied prospectively. SETTING: A department of clinical immunology investigating women with recurrent spontaneous abortions from all over Denmark. PATIENTS: Eighty-nine patients with unexplained recurrent spontaneous abortions whose pregnancies had been completed during the course of the trial. INTERVENTIONS: After randomization, 44 patients were actively immunized with husband's or third party leukocytes, and 27 patients received placebo. Eighteen patients received anticoagulation therapy in pregnancy. MAIN OUTCOME MEASURES: Changes in levels of immunoglobulin (Ig)M class and IgG class anticardiolipin antibodies after active immunization. Frequency of new miscarriages in patients who were positive or negative for anticardiolipin antibodies. RESULTS: Neither IgM nor IgG anticardiolipin antibodies changed significantly after active immunization (P greater than 0.2). The interim results of the immunization trial showed a success rate of 68% in the treated group versus 56% in the placebo group (not significantly different). Relative risk of miscarriage in anticardiolipin antibody-positive patients compared with anticardiolipin antibody-negative patients was 1.3 (95% confidence interval 0.7 to 2.2; P = 0.4) in the combined study groups. CONCLUSIONS: Patients eligible for active immunization did not exhibit significant changes in anticardiolipin antibody levels subsequent to the treatment. The treatment did not seem to provide any overall benefit with respect to pregnancy outcome. Prospectively, the risk of miscarriage in patients positive for anticardiolipin antibodies was not significantly increased.

Bioactive human recombinant tumor necrosis factor-alpha: an unstable dimer?

As determined by native polyacrylamide gel electrophoresis (PAGE) and gel chromatography the molecular mass of native tumor necrosis factor (TNF)-alpha was approximately 35 kDa. When incubated at low concentrations (less than 1 nM) 125I-labeled TNF-alpha and unlabeled TNF-alpha rapidly multimerized or dissociated into monomers and bioactivity decreased. Sodium dodecyl sulfate (SDS)-PAGE analysis of cross-linked 125I-labeled TNF-alpha demonstrated bands of multi- and trimeric TNF-alpha in addition to dominating bands of dimers and monomers. Tri-, di- and monomeric TNF-alpha were recovered from SDS-PAGE gels and allowed to renature. Of the original receptor-binding activity, 10%-15% was obtained with cross-linked TNF-alpha dimers, whereas none was recovered from preparations of trimeric TNF-alpha. Multimeric and monomeric TNF-alpha exhibited little or no binding activity, and cell-bound, cross-linked TNF-alpha which was dissociated from cellular binding sites was mainly dimeric. 125I-labeled TNF-alpha bound to lymphokine-activated killer (LAK) cells and binding kinetics were much similar (Kd approximately 100 pM) to those reported in other normal cell types. The number of receptors per LAK cell was approximately 4 x 10(3). Cross-linking of TNF-alpha to binding sites in U-937 and LAK cells yielded a receptor-ligand complex of about 80/90 kDa. At 37 degrees C, 125I-labeled TNF-alpha was rapidly internalized and degraded in L-929, U-937 and LAK cells. Degradation of ligand and recycling of receptors were blocked in the presence of methylamine. Methylamine significantly inhibited TNF-alpha-mediated cytolysis of L-929 cells and caused a quantitatively corresponding reduction in cellular TNF-alpha uptake, indicating that L-929 lysis was mediated by receptors.

Cyclosporin A mediated immunosuppression in vitro: effect on high affinity interleukin-2 receptor expression and -turnover.

The effect of in vitro administration of Cyclosporin A (CsA) during mitogen, antigen and alloantigen activation of human T-lymphocytes on high affinity interleukin-2 (IL-2) receptor expression and -turnover and IL-2 production was investigated. The presence of CsA reduced 3H-thymidine incorporation and binding of radiolabelled human recombinant (ala125) IL-2 to high-affinity receptors in a dose-dependent fashion, although a pronounced inter- and intra-individual variation in sensitivity to CsA mediated immunosuppression was observed. Maximum inhibition was obtained when antigen and CsA were added to culture medium simultaneously. Preincubation with CsA did not influence the response. Although the number of IL-2 receptors was reduced, the turnover of the remaining high affinity IL-2 receptors on CsA treated cells was unaffected. Thus, binding, internalization and degradation were qualitatively unaltered by CsA administration. Finally, T cell activation in the presence of CsA reduced radioimmuno detectable IL-2 in cell culture supernatants to about 20%. CsA, added during antigen activation, reduced the number of Tac antigen presenting cells, but anti-Tac was unable to detect variations in the expression of high affinity IL-2 receptors. The present data indicate that CsA mediates immunosuppression by affecting early events during T-cell activation, and that variations in high affinity IL-2 receptor expression and IL-2 production are secondary to this affection.

Synthesis of factor VIII in human hepatocytes in culture.

Although several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII:Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165-250 mU of VIII:Ag/10(6) cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII:Ag polypeptides as judged from the VIII:Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII:Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78-79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20-80 mU of VIII:Ag per 1 x 10(6) cells per 24 hours. Further, a significant secretion of VIII:Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII:Ag. In all of these cell cultures, vWf:Ag was indetectable or present as trace.(ABSTRACT TRUNCATED AT 250 WORDS)

Human hepatocytes in culture synthesize and secrete fibronectin.

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. Hepatocytes were cultivated in serum-free medium or 10% fetal calf serum medium supplemented with insulin, glucagon and dexamethasone. The cells were kept in culture for up to 16 days and 75% of the medium was regularly changed. Fibronectin in culture medium was detected by means of an ELISA with an assay range of 2.2-560 micrograms/l. The interassay imprecision was 6.3% at 500 micrograms/l and 14.3% at 10 micrograms/l. Significant amounts of fibronectin were detected in all cultures. During culture, fibronectin accumulated in the medium and the quantity secreted by hepatocytes by far exceeded the amounts of fibronectin associated with hepatocytes prior to cultivation. Maximum secretion rate by 10(6) hepatocytes was 167.5 +/- 73.3 ng fibronectin (mean +/- SEM, n = 3) in 24 h. When analysed by means of SDS-PAGE and immunoblotting the fibronectin isolated from hepatocyte culture medium and cell lysate co-migrated with fibronectin obtained from plasma. Our data show, for the first time, that human hepatocytes synthesize and secrete fibronectin, and it is suggested that the human liver is an important source of plasma fibronectin.

Synthesis and secretion of alpha 2-macroglobulin by human hepatocytes in culture.

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. The cells were incubated in serum-free medium or 10% FCS-medium supplemented with insulin, glucagon and dexamethasone, and kept in culture for more than 2 weeks. Seventy-five per cent of the medium was changed regularly and assayed for alpha 2-macroglobulin (alpha 2-M), pregnancy zone protein, alpha 1-antitrypsin and albumin by means of ELISA. Significant amounts of alpha 2-macroglobulin were present in all cultures. During incubation, alpha 2-M accumulated in the medium and the quantity of alpha 2-M released from the cells by far exceeded protein associated with hepatocytes prior to incubation. In 24 h 10(6) hepatocytes secreted 160.5 +/- 82.2 ng of alpha 2-M (mean +/- SD, n = 5). Cell-associated, as well as secreted alpha 2-M appeared to be on native form, as determined by immunoisolates from lysed cells and culture supernatants. Pregnancy zone protein was only detected in about 50% of the cultures and its rate of secretion was less than 2 ng 24 h-1 per 10(6) cells. In contrast, culture medium contained considerable quantities of alpha 1-antitrypsin and albumin. In 24 h, 10(6) hepatocytes released greater than 2 micrograms alpha 1-antitrypsin and greater than 5 micrograms albumin. The present study suggests the hepatocyte to be of major importance for the synthesis of intravascular alpha 2-M.

Human hepatocytes exhibit receptors for alpha 2-macroglobulin and pregnancy zone protein-proteinase complexes.

Hepatocytes were isolated by application of the two-step collagenase technique to pieces of human liver. 125I-labelled alpha 2-macroglobulin-trypsin complex bound to hepatocytes at 4 degrees C with a half time of approximately 4.5 h. At near equilibrium half of the receptors were saturated at an alpha 2-macroglobulin-trypsin complex concentration of about 60 pmol 1(-1) and the Scatchard plot was linear. Dissociation of the labelled complex was slow (T1/2 = 24 h) at low receptor occupancies. At high receptor occupancies dissociation was biphasic with a rate constant (K-1) for the initial rapid phase of about 2.4 x 10(-2) min-1. Labelled alpha 2-macroglobulin-trypsin complex bound at 4 degrees C was rapidly internalized at 37 degrees C (T1/2 = 1.9 min), and in 3.5 h approximately 10% of the label was released into the medium in a trichloroacetic acid-soluble form. At 37 degrees C, 125I alpha 2-macroglobulin-trypsin was taken up by hepatocytes and trichloroacetic acid soluble radioactivity appeared in the medium following a sigmoidal curve. Similar results were obtained with 125I-pregnancy zone protein-chymotrypsin complex. At 4 degrees C, hepatocytes bound nearly equal amounts of labelled alpha 2-macroglobulin-trypsin and pregnancy zone protein-chymotrypsin complex, and a large excess (100 nmol 1(-1) of one of the macroglobulins could almost completely abolish binding of trace amounts (5-20 pmol 1(-1] of the other. The present findings strongly suggest that the hepatocyte is of major importance for removal of alpha 2-macroglobulin- and pregnancy zone protein-proteinase complex in humans, in agreement with previous results in rats and mice.

Binding, uptake and degradation of human recombinant interleukin-2 (125 ala) in activated human T- and B-lymphocytes and in monocyte-macrophages.

High-affinity receptors for IL-2 (ala 125) were demonstrated in PHA-, antigen- and/or alloantigen-activated human T-cells (both proliferative and cytotoxic), in PWM-activated human B-cells and in human monocyte-macrophages. Binding in PHA-blasts was irreversible and Ca++-independent, and labelled IL-2 (ala 125) bound at 4 degrees C could not be removed by trypsin treatment. Binding was strongly pH-dependent, and lowering of pH caused release of nearly all cell associated radioactivity at 4 degrees C. In T- and B-lymphocytes, additional binding at high ligand concentrations was accounted for by receptors of much lower affinity. Binding to low-affinity receptors appeared reversible. At 4 degrees C, 2.2 pM labelled IL-2 (ala 125) bound to PHA-blasts (3.6 X 10(6)/ml) with a half time of about 15 min, and the association rate constant was approximately 8 X 10(9) M-1 min-1. The number of high affinity receptors per T-cell was determined as 9.7 +/- 0.5 X 10(2). At 37 degrees C, 60% of the tracer bound at 4 degrees C was rapidly internalized (Kint = 0.89 X 10(-1) min-1), and radioactivity comprising small MW products and iodotyrosine was released following a sigmoidal curve after a 20 min lag period. Similar results were obtained in PWM-activated B-lymphocytes and in cultured monocytes. It is concluded that high-affinity receptors mediate binding, uptake and degradation of IL-2 in activated human T- and B-lymphocytes and in monocyte-macrophages.