Primary pleural thymoma with coexistent incidental small hepatocellular carcinoma - an autopsy case report with brief review of literature.

Primary pleural thymomas are rare tumors often mistaken for malignant mesothelioma clinically and radiologically. An autopsy case report of primary pleural thymoma associated with a coincidental small hepatocellular carcinoma is presented. This case is reported because of the rarity of pleural thymoma and the coincidental finding of a small hepatocellular carcinoma in a non-cirrhotic background. The literature on these two tumors has been reviewed.

Evidence of age-related changes in the antennal glomeruli of Drosophila melanogaster using monoclonal antibodies.

As a tool to better understand the organization of the olfactory pathway three monoclonal antibodies have been isolated and characterized each having a unique staining pattern in the antenna and antennal lobe of Drosophila melanogaster. Monoclonal antibody F14-2D6 stains sensilla coeloconica and thick sensilla basiconica in the funiculus, Y1-3D10 stains only a few sensilla especially in and around the sacculus, while F15-12E8 stains all the sensilla. All three antibodies stain a subset of the glomeruli in the antennal lobe, of which 11 glomeruli are stained in common by all three antibodies. These antibodies could be used to study projection patterns of the sensilla into the antennal lobe. Glomerular staining was observed at different developmental times with the different antibodies. F15-12E8 stains all the glomeruli at eclosion, Y1-3D10 stains only a few glomeruli at eclosion but most glomeruli are stained by the first day after eclosion. F14-2D6 stains all glomeruli only after eclosion. F15-12E8 also stains the mushroom bodies. The antigen recognized by F14-2D6 in the glomeruli shows an increase with age of the flies, measured as increased intensity of staining. These observations suggest that age-related changes continue in the antennal lobe of the flies even after eclosion. These antibodies could therefore serve as unique markers for other studies on the development of the olfactory system.

Rat interleukin 6: expression in recombinant Escherichia coli, purification and development of a novel ELISA.

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS).

Biological activity and brain actions of recombinant rat interleukin-1alpha and interleukin-1beta.

IL-1alpha and IL-1beta have potent effects on the central nervous system resulting in fever, activation of the hypothalamic-pituitary-adrenal axis and behavioural depression. These effects have mainly been studied in rats, using recombinant human and mouse IL-1. Because IL-1alpha and IL-1beta show some species specificity in the potency of their biological activities, the objective of the present work was to directly compare the effects of recombinant rat IL-1alpha and IL-1beta in the rat system as a first step to dissect out the mechanisms that are involved in these effects. In vitro, recombinant rat IL-1alpha and IL-1beta bound with the same affinity as human IL-1 to the rat insulinoma Rin m5F cell line that mainly expresses type I IL-1 receptors. This binding activated IL-1 receptors, as shown by induction of the synthesis of TNF-alpha mRNA. In vivo, recombinant rat IL-1alpha and IL-1beta enhanced body temperature, increased plasma levels of corticosterone and ACTH, and depressed social behaviour. All these effects were obtained at doses 100-1,000 fold lower when IL-1 was injected centrally than when it was administered peripherally, indicating that they are centrally mediated. The relative potencies of recombinant rat IL-1alpha and IL-1beta were not the same depending on the endpoint and the route of injection, indicating that different mechanisms are likely to be involved in the various effects of IL-1 on the brain.

Lectin-binding assays for the isoforms of human erythropoietin: comparison of urinary and four recombinant erythropoietins.

Assays have been developed for the isoforms of erythropoietin (EPO) based on their binding to eight different lectins. These assays were used to compare the isoform compositions of two preparations of human urinary EPO (uEPO) and four preparations of recombinant DNA-derived human EPO (rEPO), which had been shown to differ in their biological and immunological properties and in their isoform composition as judged by isoelectric focusing and electrophoresis. Agarose-bound Ricinus communis agglutinin I (RCA), Erythrina cristagalli agglutinin (ECA), Maackia amurensis leukoagglutinin (MAL), Sambucus nigra agglutinin (SNA), Lycopersicon esculentum agglutinin (LEA), concanavalin A (Con A), Phaseolus vulgaris agglutinin-L4 (L-PHA) and Agaricus bisporus agglutinin (ABA) were used to bind EPO isoforms possessing: N-glycans containing non-sialylated outer Gal beta 1-4GlcNAc (RCA and ECA), NeuAc alpha 2-3Gal beta 1-4GlcNAc (MAL), NeuAc alpha 2-6Gal (SNA), or repeating Gal beta 1-4GlcNAc sequences (LEA); biantennary N-glycans (Con A); tetraantennary and 2,6-branched triantennary N-glycans (L-PHA); and O-glycans containing NeuAc alpha 2-6GalNAc (SNA) and Gal beta 1-3GalNAc (ABA). Free EPO was measured by mouse spleen cell bioassay or immunoassay. Estimates from most lectin-binding assays were reproducible between assays and batches of lectin-agarose, although batches of MAL- and ABA-agarose, and to a lesser extent LEA-agarose, differed in their EPO-binding. Lectin-binding assays showed differences between the isoform compositions of all EPOs, including the two Chinese hamster ovary cell-derived rEPOs, with RCA- and ECA-binding assays being the most discriminating. Lectin-binding estimates provided evidence that uEPO differs from these rEPOs in its lower content of isoforms with biantennary N-glycans and higher content of those with multiantennary N-glycans, and in its lower content of isoforms with N-glycans possessing repeating Gal beta 1-4GlcNAc sequences and of those with O-glycans containing Gal beta 1-3GalNAc. Lectin-binding estimates also indicated that, contrary to some reports, uEPO possesses Gal beta 1-3GalNAc-containing O-glycans but not NeuAc alpha 2-6GalNAc-containing O-glycans or NeuAc alpha 2-6Gal-containing N-glycans. Most groups of lectin-bound EPO isoforms did not differ in their relative bioactivities and immunoreactivities. However, estimates for ABA-bound EPO isoforms suggested that O-glycans might influence the bioactivity of EPO differently to its immunoreactivity. Furthermore, the bioactivities of some ECA-bound EPO isoforms were higher, and those of some of the MAL-bound EPO isoforms lower, than their immunoreactivities, consistent with the reported enhancement of EPO in vitro bioactivity by desialylation.

Inhibition of tissue factor and cytokine release.

The effect of inhibitors of cytokine release and plasma coagulation on lipopolysaccharide (LPS)-induced tissue factor and interleukin-6 (IL-6) was investigated. Dexamethasone, an inhibitor of cytokine production, inhibited LPS-induced tissue factor and IL-6 release by mononuclear cells (MNC), but enhanced IL-1beta-evoked tissue factor activity. Clinical antithrombin (AT) concentrates inhibited, in a dose-dependent manner, tissue factor and IL-6 production by MNC and human umbilical vein endothelial cells (HUVEC). The three AT preparations tested, when compared using the same antithrombin unit, had different potencies. Activated protein C (APC) augmented LPS stimulation of HUVEC and further increased the production of tissue factor and IL-6. The same effect was not observed with MNC; LPS-induced tissue factor and IL-6 release were unaffected by APC. Truncated tissue factor pathway inhibitor (TFPI1-161) inhibited LPS-induced MNC tissue factor and IL-6 production, but was unable to prevent LPS stimulatory activity on HUVEC. These data suggest a complex interaction between the coagulation pathway and the cytokine network.

hsp70 synthesis in Schwann cells in response to heat shock and infection with Mycobacterium leprae.

Induction of heat shock protein synthesis was monitored in murine and monkey Schwann cells exposed to elevated temperatures. Synthesis of the stress-inducible 70-kDa heat shock protein (hsp70) was detected in both murine and primate Schwann cells by metabolic labelling and by immunoblotting with a specific monoclonal antibody. hsp70 synthesis was also induced in Schwann cells after infection with Mycobacterium leprae and was detected from 24 h to 1 week postinfection. These results are discussed with respect to the possible role of heat shock proteins in immunopathological events associated with the clinical manifestations of leprosy.

Radiolabeling of Mycobacterium leprae lipids within schwannoma cells, a potential drug screening system.

This study describes a novel method which could be developed into a test system of evaluating the efficacy of antileprosy drugs. The method estimates incorporation of [14C]acetate into lipids of Mycobacterium leprae maintained within the 33B Schwannoma cell line. Schwannoma cell-resident M. leprae cells incorporated significant levels of radiolabel within their lipids during 12 days of incubation in vitro. This incorporation was markedly reduced by 5 micrograms of rifampin per ml (decrease, 81.62%); this decrease was observed within 24 h of addition of the drug. Dapsone also reduced the radiolabel incorporation into the lipids, but to a lesser extent (decrease, 27.58%). This system was also able to differentiate between rifampin-sensitive and -resistant strains of mycobacteria. It is suggested that since the effect of bacteriostatic (dapsone) and bactericidal (rifampin) drugs could be detected by using this technique, it may prove useful in screening novel drugs acting against M. leprae.

Plasma clearance in the rat of the LH bioactivity of two human LH standards of differing molecular composition.

The LH biological potency of the International Reference Preparation (IRP) of Human Pituitary LH for Immunoassay (IRP 68/40) relative to that of the 2nd IRP of Human Pituitary FSH and LH for Bioassay (IRP 78/549) is markedly greater when estimated by in-vitro interstitial cell testosterone production (TICT) bioassay than by in-vivo bioassay, and by the 4-h ovarian ascorbate depletion (OAAD) assay than by the 4-day seminal vesicle weight gain assay. Other preparations of human LH which, like IRP 68/40, were highly purified, showed a similar spectrum of bioactivity in these assay systems and also contained a higher proportion of more basic LH isoforms than are found in crude pituitary extracts such as IRP 78/549. In an attempt to explain these differences, a comparison was made of the plasma survival in rats of the LH bioactivity (by TICT assay) of these two preparations. Contrary to expectation, their relative plasma clearance rates over a 4-h period did not account for their differing bioactivities. The plasma half-life of the LH bioactivity (with 95% confidence limits) was estimated to be 42.4 (35.3-49.5) min for IRP 68/40 and 41.3 (31.5-51.0) min for IRP 78/549. Furthermore the time-course of action in vivo of IRP 78/549 did not appear to be more prolonged than that of IRP 68/40. Thus their plasma testosterone responses during the course of these 4-h plasma clearance studies were similar, and estimates of the LH potency of IRP 68/40 relative to that of IRP 78/549 were no greater by 2-h than by 4-h OAAD assay.(ABSTRACT TRUNCATED AT 250 WORDS)