IL-6 produced by Kupffer cells induces STAT protein activation in hepatocytes early during the course of systemic listerial infections

Kupffer cells were the principal source of IL-6 produced in the livers of mice following i.v. inoculation of Listeria monocytogenes. IL-6 mRNA expression and the production of IL-6 were reduced drastically within the nonparenchymal liver cell population derived from mice rendered Kupffer cell depleted by pretreatment with liposome-encapsulated dichloromethylene diphosphonate. A sharp increase in the appearance of activated STAT3 occurred in extracts of purified hepatocytes derived from normal mice infected i.v. with Listeria. Remarkably, the kinetics of this increase overlapped IL-6 mRNA expression by Kupffer cells; each peaked at approximately 30 min postinfection. No increase in STAT3 activation was observed in IL-6-deficient or Kupffer cell-depleted animals. The results of these experiments indicate that the synthesis of IL-6 and the activation of STAT3 within hepatocytes are critical functions of Kupffer cells occurring very early during the course of systemic listerial infections.     

Decay-accelerating factor is a component of subendothelial extracellular matrix in vitro, and is augmented by activation of endothelial protein kinase C

The vasculature is protected from complement activation by regulatory molecules expressed on endothelial cells. However, complement fixation also occurs on subendothelial extracellular matrix (ECM) in vitro, and is initiated simply by retraction or removal of overlying cells. To investigate mechanisms controlling vascular complement activation, we examined subendothelial ECM for the presence of complement regulatory proteins. Decay-accelerating factor (DAF) was found on both human umbilical vein endothelial cells (HUVEC) and in their ECM; in contrast, membrane cofactor protein was found only on cells. ECM and HUVEC DAF were distinguishable based on several properties. While HUVEC DAF is anchored to cell membranes by a phospholipase C-sensitive glycosylphosphatidylinositol linkage. DAF was removed from ECM only by proteolytic digestion. Cytokines (TNF-alpha, IL-1 beta, IL-4) increased HUVEC DAF expression, but had minimal effect on ECM DAF; in contrast, phorbol 12-myristate 13-acetate (PMA) and wheat germ agglutinin markedly increased DAF on both HUVEC and ECM. The effect of PMA was mediated by activation of protein kinase C. The complement regulatory potential of ECM DAF was assessed by evaluating the effect of DAF-neutralizing antibodies on C3 deposition on HUVEC ECM, as well as on HeLa cell ECM, which had a considerably higher DAF content. DAF blockade enhanced C3 deposition on HeLa ECM, but had no effect on HUVEC ECM. As ECM DAF is likely to be immobile, i.e. able to interact only with C3 convertases forming in the immediate vicinity, its ability to regulate complement activation may be particularly density dependent, and contingent on endothelial-dependent up-regulation.     

Role of beta-adrenoceptor on renal interleukin-6 and tumor necrosis factor in spontaneously hypertensive rats

We have shown previously in the spontaneously hypertensive rat (SHR) kidney that interleukin-6 (IL-6) and tumor necrosis factor (TNF) mRNA levels were low under conditions of acute anaesthesia and surgical stress. The reasons for the suppression of IL-6 and TNF gene expression in the SHR were investigated by examining the influence of enhanced beta-adrenergic stimulation, high blood pressure, and renal function (renal blood flow, glomerular filtration rate, plasma creatinine levels) on renal IL-6 and TNF mRNAs. The experiments were performed by means of the following three studies; (1) SHR and Wistar rats at 4, 7, 9 week old were injected with lipopolysaccaride (LPS), and then a relationship between blood pressure levels and IL-6 and TNF mRNA levels were estimated, (2) isoproterenol and propranolol were administered into SHR and WKY rats, and the levels of IL-6 and TNF mRNA were compared, (3) under condition of anaesthesia and surgical stress, blood pressure and renal functions in SHR were measured, and then the relationships between these factors and IL-6 or TNF mRNA levels were analyzed. Renal IL-6 and TNF mRNAs in SHR remained low even though blood pressure increased with age and there was no significant correlation between IL-6 or TNF mRNA levels and values of blood pressure or renal function under anaesthesia and surgical stress. However, the inhibition of the IL-6 and TNF mRNAs in SHR was prevented by propranolol treatment. These results suggested that suppression of IL-6 and TNF mRNAs in the SHR kidney could be due to overactivity of beta-adrenergic influences which may importantly contribute to the development of hypertension.     

Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.     

Circuit-specific alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys

Age-associated memory impairment occurs frequently in primates. Based on the established importance of both the perforant path and N-methyl-D-aspartate (NMDA) receptors in memory formation, we investigated the glutamate receptor distribution and immunofluorescence intensity within the dentate gyrus of juvenile, adult, and aged macaque monkeys with the combined use of subunit-specific antibodies and quantitative confocal laser scanning microscopy. Here we demonstrate that aged monkeys, compared to adult monkeys, exhibit a 30.6% decrease in the ratio of NMDA receptor subunit 1 (NMDAR1) immunofluorescence intensity within the distal dendrites of the dentate gyrus granule cells, which receive the perforant path input from the entorhinal cortex, relative to the proximal dendrites, which receive an intrinsic excitatory input from the dentate hilus. The intradendritic alteration in NMDAR1 immunofluorescence occurs without a similar alteration of non-NMDA receptor subunits. Further analyses using synaptophysin as a reflection of total synaptic density and microtubule-associated protein 2 as a dendritic structural marker demonstrated no significant difference in staining intensity or area across the molecular layer in aged animals compared to the younger animals. These findings suggest that, in aged monkeys, a circuit-specific alteration in the intradendritic concentration of NMDAR1 occurs without concomitant gross structural changes in dendritic morphology or a significant change in the total synaptic density across the molecular layer. This alteration in the NMDA receptor-mediated input to the hippocampus from the entorhinal cortex may represent a molecular/cellular substrate for age-associated memory impairments.     

Plasma activity of contact coagulation factors in patients with IgA nephritis

A cellular electrical impedance device which can detect the activated state of eosinophils has been developed and tested. This impedance device consists of a small gold electrode (50 microns x 50 microns) and a large gold electrode (1.5 cm x 0.5 cm) on a glass substrate, and it was fabricated by standard photolithographic techniques. Eosinophils, which belong to the granulocytic class of white blood cells, exhibit different physical properties when they change from the nonactivated state to the activated state. Hypothetically, these changes should correspond to a change in the measured electrical impedance. In this paper, data from the measured electrical impedance of eosinophils is presented. The measurements show that the average impedance of the activated eosinophils is 26% lower than the average impedance of the nonactivated eosinophils. Statistical analysis of the measured data shows that there is a significant difference between the measured impedances of activated and nonactivated eosinophils.