Tumor therapy with an antibody-targeted superantigen generates a dichotomy between local and systemic immune responses.

Repeated injections of a fusion protein containing the superantigen staphylococcal enterotoxin A (SEA) combined with a Fab fragment of a tumor-specific antibody is a highly efficient immunotherapy for mice expressing lung melanoma micrometastasis. In the present study, the systemic and local immune responses generated by this therapy were analyzed at a cellular level. Two distinct but coupled immune reactions occurred after repeated therapy. Tumor necrosis factor and macrophage inflammatory protein-1 alpha and -1 beta were immediately synthesized, in the absence of T lymphocytes, at the local tumor site in the lung. This was followed by the induction of VCAM-1 adhesion molecule expression on pulmonary vascular endothelial cells. Concurrently, the early response in the spleen was characterized by the induction of selective T cells producing interleukin (IL)-2. The primed and expanded SEA-reactive V beta 3- and V beta 11-expressing T lymphocytes accumulated to the tumor area only after Fab-SEA therapy and were not present in the lung when SEA, Fab fragment, or recombinant IL-2 was injected. The tumor-infiltrating T cells produced large amounts of interferon-gamma, but no IL-2 or Th2 type of lymphokines were detected at the tumor site in the Fab-SEA-targeted antitumor immune response. These results emphasize the necessity to investigate several sites of antigen presentation to elucidate the effects of immunotherapy.     

Innate immunity, macrophage activation, and atherosclerosis

Summary:  Inflammation underpins the development of atherosclerosis. Initiation and progression of vascular inflammation involves a complex cellular network, with macrophages as major contributors. Activated macrophages produce proinflammatory mediators, bridge innate and adaptive immunity, regulate lipid retention, and participate directly in vascular repair and remodeling. Recent efforts to elucidate molecular mechanisms involved in the regulation of vascular inflammation in atherosclerosis have implicated several families of innate immune recognition receptors in inflammatory activation during the course of this disease. This article reviews our current understanding of innate immune recognition receptors, signaling pathways, and putative ligands implicated in activation of macrophages in the disease. In its final section, we propose a model for the role of macrophages in bridging inflammation and atherosclerosis from the perspective of innate immune recognition and activation.     

Effect of a new synthetic serum replacement on insulin and somatostatin secretion from isolated rat pancreatic islets in long-term culture

Summary Serum contains insulin degrading components. We have evaluated the insulin and somatostatin secretion from isolated rat pancreatic islets during a 2-wk culture period using three different serum-containing media, and one serum-free medium with a synthetic serum replacement. Islets incubated in serum-free medium elicited significantly higher daily insulin and somatostatin secretions than islets incubated in the serum-containing media. After a 2-wk culture period, islets from the serum-free medium secreted significantly more insulin and somatostatin than islets cultured in other media when stimulated with 25 mmol/liter glucose together with 15 mmol/liter theophylline. We conclude that the serum-free medium is superior for long-term culture of rat pancreatic islets.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

Evidence for cell death in the vascular endothelium in vivo and in vitro

Focal, spontaneous cell death in the rat aortic endothelium was demonstrated by cytochemistry. Cells with intracellular calcium deposits, indicating cell death with mitochondrial calcification, were identified by chlorotetracycline fluorescence. The same cells also contained cytoplasmic IgG, which binds to cytoskeletal components of the dead cell. The immunocytochemical detection of IgG in en face preparations was used as a quantitative method for detecting cell death in the aortic endothelium. The use of an indirect immunoperoxidase technique and "Häutchens" of paraformaldehyde-fixed tissue provided high sensitivity and cellular recovery with low background. A cell death frequency of 0.19% +/- 0.04% was observed in 5-month-old Sprague-Dawley rats. When compared with the replication rate of aortic endothelium in these animals, the data suggest that dead cells remain in the endothelium for more than 24 hours. This conclusion was supported by in vitro studies. Confluent cultures of bovine aortic endothelium were pulsed with trypan blue, and the residence time of blue cells was 3.5-4 days in non-flow culture system. Time-lapse video microscopy showed a prolonged cell death process with a phase of rapid intracellular movements, followed by undermining by surrounding cells and fragmentation of the dead cell. Migration of surrounding cells rapidly covered partial detachments of the dead cell, so that no holes could be detected in the monolayer when the dead cell finally detached. It is concluded that the normal turnover of cells in the aortic endothelium involves a prolonged phase of in situ cell death and finally detachment with very little or no exposure of subendothelial structures.     

Introduction of a new hepatitis agent in retrospect: genetic studies of Swedish hepatitis D virus strains.

The supposedly first outbreak of hepatitis D virus (HDV) infection in Sweden occurred among intravenous drug addicts in the Malmö area in the mid-1970s. Stored sera from this outbreak were used for viral RNA extraction and analysis. By sequence comparisons, the HDV genomes from those Swedish patients fell into two separate clusters, within which the RNA sequences were closely related. These two HDV groups genetically resembled the French and US-1 isolates of genotype I, respectively, indicating that there had been at least two separate sources of HDV infection. The genetic alterations of the HDV RNA were investigated by sequence analysis of nine annually drawn serum samples from one patient and paired samples collected between 2 and more than 10 years apart from six patients with chronic HDV infection. Only mutational changes were observed, and no insertion or deletion appeared throughout the periods observed. It was found that the Swedish HDV isolates mutationally evolved at an average rate of 1.1 x 10(-3) substitutions per nucleotide per year over a long time course of chronic HDV infection, which is of the same magnitude as that of other RNA viruses.     

Effects of subacute treatment with toluene on cerebrocortical alpha- and beta-adrenergic receptors in the rat. Evidence for an increased number and a reduced affinity of beta-adrenergic receptors

Subacute treatment with toluene (80-1500 p.p.m.) produces a dose-dependent reduction of affinity and increase in density of the beta-adrenergic antagonist [3H]dihydroalprenolol binding sites in the frontoparietal cortex of the male rat, while the binding characteristics of alpha 1-adrenergic ([3H]WB 4101) and alpha 2-adrenergic ([3H]p-aminoclonidine) binding sites in the same region is unaffected by this treatment as evaluated in vitro. Therefore, it is suggested that the cortical beta-adrenergic receptors are particularly vulnerable to the action of toluene in vivo. It is speculated that as a result cortical beta-adrenergic neurotransmission may be altered following exposure to low concentrations of toluene, possibly related to the physico-chemical properties of toluene, leading to changes in membrane fluidity.     

Analogue simulator of e.e.g. signals based on spectral components

A model describing the spectral properties of e.e.g signals by a set of parameters has previously been introduced. It leads to a decomposition of the spectrum into one or more spectral components. The described e.e.g.-signal simulator is based on such a decomposition into one delta, and three rhythmic components called theta, alpha and beta denoting the location of the resonance peaks. Each component is generated separately with a noise generator and a spectral shaping filter. Care is taken to make the noise generator have a flat spectrum down to zero frequency. The spectral shaping filters are of an active type with a configuration that allows easy adjustment of poles and zeros. The use of the equipment is demonstrated by simulating five different e.e.g. signals first by analysing the e.e.g., and then by setting the simulator to produce an identical spectrum.     

Influence of sodium deoxycholate on morphology, net fluid transport and motility in the small intestine of the rat

Intestinal fluid secretion and motility were induced by luminal perfusion of rat small intestine with sodium deoxycholate, a dihydroxy bile salt for 1-3 h. Changes in intestinal morphology were studied simultaneously with the changes in fluid transport and motility. The results suggest that the bile salt causes epithelial lesions which may lead to a reduced fluid absorption in the villi, thereby explaining part of the total change in net fluid transport caused by the bile salt. Pyrilamine and indomethacin did not influence the bile salt-induced secretion. Based on earlier studies, it is proposed that the major part of the bile salt-evoked secretion is mediated via activation of intramural nervous reflex(es), which also stimulate the intestinal smooth muscle cells.