Activation of microglia and astrocytes by CpG oligodeoxynucleotides.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate cells of the immune system to secrete a variety of cytokines and chemokines. This function can be carried out by microglia and astrocytes in the CNS. To evaluate the effect of CpG ODN on microglia and astrocytes, purified cells were isolated and cultured in vitro. CpG ODN rapidly up-regulated their production of IL-1beta, IL-6, IL-12, TNFalpha, MIP-1alpha and/or MIP-1beta. In vivo, systemically administered CpG ODN up-regulated the expression of mRNA encoding cytokines and chemokines in normal mouse brain. These findings suggest that CpG ODN can directly activate immune cells of the CNS.

Differences in kinetics of human cytomegalovirus cell-free viral release after in vitro infection of human microglial cells, astrocytes and monocyte-derived macrophages.

Microglial cells and astrocytes isolated from human embryonic proencephalon were compared to monocyte-derived macrophages (MDM) for their ability to replicate human cytomegalovirus (HCMV) in vitro. A specific cytopathic effect was observed in microglial cells and astrocytes, but not in MDM. A high percentage of glial cells but a low percentage of MDM expressed immediate-early and late viral antigens. The ability of HCMV-infected microglial cells and astrocytes to release viral particles in their supernatants was significantly higher than that of infected MDM. Human microglial cells and astrocytes at an early stage of development are highly susceptible to HCMV infection.

Negative feedback between prostaglandin and alpha- and beta-chemokine synthesis in human microglial cells and astrocytes.

The understanding of immune surveillance and inflammation regulation in cerebral tissue is essential in the therapy of neuroimmunological disorders. We demonstrate here that primary human glial cells were able to produce alpha- and beta-chemokines (IL-8 > growth related protein alpha (GROalpha) >> RANTES > microphage inflammatory protein (MIP)-1alpha and MIP-1beta) in parallel to PGs (PGE2 and PGF2alpha) after proinflammatory cytokine stimulation: TNF-alpha + IL-1beta induced all except RANTES, which was induced by TNF-alpha + IFN-gamma. Purified cultures of astrocytes and microglia were also induced by the same combination of cytokines, to produce all these mediators except MIP-1alpha and MIP-1beta, which were produced predominantly by astrocytes. The inhibition of PG production by indomethacin led to a 37-60% increase in RANTES, MIP-1alpha, and MIP-1beta but not in GROalpha and IL-8 secretion. In contrast, inhibition of IL-8 and GRO activities using neutralizing Abs resulted in a specific 6-fold increase in PGE2 but not in PGF2alpha production by stimulated microglial cells and astrocytes, whereas Abs to beta-chemokines had no effect. Thus, the production of PGs in human glial cells down-regulates their beta-chemokine secretion, whereas alpha-chemokine production in these cells controls PG secretion level. These data suggest that under inflammatory conditions, the intraparenchymal production of PGs could control chemotactic gradient of beta-chemokines for an appropriate effector cell recruitment or activation. Conversely, the elevated intracerebral alpha-chemokine levels could reduce PG secretion, preventing the exacerbation of inflammation and neurotoxicity.

[Productive replication of human cytomegalovirus in primary microglial cells]. / Réplication productive du cytomégalovirus humain dans des cultures primaires de cellules microgliales.

Human cytomegalovirus (HCMV) infection can result in neurological symptoms. In vitro replication of the HCMV was studied in primary cultures of microglial cells from the central nervous systems (CNS) of human embryos. The microglial cells were infected with various amounts of either the AD169 laboratory HCMV strain or a clinical HCMV isolate. A specific cytopathic effect occurred within 24 h and persisted for two months. Immunocytochemical tests for immediate early and late viral antigens done one and three days after the infection demonstrated that 60% to 80% of the microglial cells were infected and that 3% to 8% were the site of viral DNA replication. Kinetic studies showed accumulation of viral particles in the supernatant during the first two weeks after the infection. Prestimulation of the cells by PMA 24 h before the infection was associated with increased release of viral particles and with an increased percentage of cells expressing late viral antigens. The microglial cells of the human embryonic CNS are fully permissive targets for the HCMV. The in vitro HCMV model used in this study may prove useful for investigating the pathophysiology of HCMV encephalitis, in particular after mother-to-fetus transmission of the virus.

Induction of human immunodeficiency virus type 1 replication in human glial cells after proinflammatory cytokines stimulation: effect of IFNgamma, IL1beta, and TNFalpha on differentiation and chemokine production in glial cells.

Although evidence for human immunodeficiency virus 1 (HIV-1) presence in the central nervous system (CNS) of infected patients is well established, the intensity of viral replication within the brain is not usually known. In vitro, human embryonic microglial cells internalized HIV-1 through a CD4-dependent pathway but were not permissive to viral replication. We observed that HIV replication was induced when CNS cell cultures were stimulated for 14 days by a combination of proinflammatory cytokines including IFNgamma, IL1beta, and TNFalpha. After long-term cytokine stimulation, morphologically differentiated glial cells appeared, in which HIV-1 tat antigen was detected after infection. Thus, variations in the stage of maturation/activation of CNS cells under inflammatory conditions probably play a major role in facilitating massive production of HIV-1. We then studied the effect of prolonged cytokine stimulation on the secretion of inflammatory mediators by glial cells. An early increased secretion of prostaglandin F2alpha and chemokines (RANTES>>MIP-1alpha>>MIP-1beta) was observed, due to both microglia and astrocytes. In contrast to persistent PGF2alpha production, an extinction of RANTES and MIP-1beta but not of MIP-1alpha secretion occurred during the 14 days of stimulation and was inversely correlated with the ability of glial cells to replicate HIV-1. The study of the secretory factors produced in response to a persistent inflammation could provide a better understanding of the modulation of HIV replication in glial cells.

Endogenous nitric oxide activates prostaglandin F2 alpha production in human microglial cells but not in astrocytes: a study of interactions between eicosanoids, nitric oxide, and superoxide anion (O2-) regulatory pathways.

Secretion of the eicosanoids, nitric oxide (NO.) and superoxide anion (O2.-) was evaluated in human embryonic astrocytes and microglia. An inducible form of cyclo-oxygenase (COX 2) was demonstrated in astrocytes and microglia after IL-1 beta plus IFN-gamma stimulation; since 1) large amounts of PGF2 alpha were released; 2) PGF2 alpha secretion required protein synthesis and was blocked by indomethacin; and 3) the response was delayed and persistent. Using the same inducers, astrocytes, but not microglial cells, produced NO. and had an inducible form of nitric oxide synthase. Conversely, microglial cells were induced by IL-1 beta and IFN-gamma to generate superoxide anions (O2.-) through an NADPH oxidase-dependent pathway. We then investigated interactions between these different pathways of synthesis by inhibition experiments. The cytokine-induced production of PGF2 alpha in astrocytes was not affected by exposure to N-omega-monomethyl-L-arginine, which inhibits NO. production, whereas it was reduced by 40% in microglia. Since microglia did not secrete any detectable NO. in their supernatant, intracellular production of NO. could occur in these cells that positively regulated PGF2 alpha production. Exposure to indomethacin, which prevented PGF2 alpha production in both astrocytes and microglia, resulted in a 64% increase in cytokine-induced NO. production by astrocytes and a 70% inhibition of O2.- generation by stimulated microglia. Finally, superoxide dismutase depletion of O2.- in astrocytes and microglia had no effect on PGF2 alpha production in these cells. These results demonstrate that there are important interactions between the pathways of synthesis of inflammatory mediators in glial cells that could unveil additional regulatory mechanisms.

Effect of interferon gamma and TNF alpha on the differentiation/activation of human glial cells: implication for the TNF alpha receptor 1.

This study focused on the activation/differentiation of human microglial cells and astrocytes, which is a prerequisite for HIV1 replication. In vitro, IFN gamma induced a differentiation-like morphological change in embryonic microglia and astrocytes, in both primary and in purified culture. This effect was enhanced by TNF alpha which in itself had no effect. IFN gamma did not increase the low level of endogenous TNF alpha, which is not secreted by embryonic cells, but stimulated the expression of TNF alpha R1, although to a relatively low extent. IFN gamma facilitated TNF alpha response through an increase in TNF alpha R1 expression, but probably also through interaction with different intracellular signal transduction pathways.

Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen.

Four continuous cell lines of human microglial cells were obtained by transfection of enriched cultures of human embryonic brain-derived macrophages with a plasmid encoding for the large T antigen of SV40. The transformed cells had the macrophagic characteristics of adherence and intra-cytoplasmic non-specific esterase activity. They could phagocytize zymosan particles but the phagocytic activity remained low. They expressed several macrophagic antigens but not the monocytic markers CD14, CD4, CD68/Ki-M6 and CD11c. The cells could be activated to express class II major histocompatibility complex antigens after interferon-gamma activation. Finally, interleukin-6 was produced spontaneously by the cells and this production was further increased after interleukin-1 alpha stimulation.

HIV-1 and the developing human nervous system: in vivo and in vitro aspects.

HIV-1 is able to penetrate the developing human central nervous system and induce damage. Clinical and neuropathological observations demonstrate that (a) the virus is transmitted during the late phase of pregnancy or at the time of delivery; (b) the main infected cells within the brain are macrophages, and (c) the mere presence of infected macrophages within CNS tissue is not enough to predict disease. Primary cultures of human embryonic or fetal CNS and derived cultures highly enriched in either astrocytes or microglial cells are useful tools to study the mechanisms of HIV-1-induced neuronotoxicity in which both microglial cells and astrocytes play important roles.

Effect of copper-containing intrauterine contraceptive devices (IUCD) on growth of endometrial and cervical cells.

PIP: Physicians at the School of Medicine at the University of Baghdad in Iraq took a biopsy of endometrial and cervical tissue from women between 25-40 years old before and after using a copper IUD. Researchers exposed each tissue type to autoradiography using 5% methyl tritiated thymidine to indicate active cell metabolism. They also examined each type with an electron microscope. Thymidine uptake fell as duration of a copper IUD was used. For example, 52% of the endometrial and cervical cells were viable before insertion of the copper IUD. After 5 months, thymidine uptake fell remarkably to 32%. It fell to 20% at 12 months, 10% at 18 months, and 6% at 36 months. The thymidine uptake between endometrial and cervical tissue was not statistically different. The major change in the cervix as observed under electron microscopy included a few to a significant number of lymphocytes between the epithelial cells. Apical protrusions remained the same in the secretory cells of the endothelial lining in the endometrium and the glands. In tissues exposed to a copper IUD, the mitochondria of cells in the lumen and glands swelled and the number of lysosomes increased. Further, the number of lymphocytes also increased in both the cervix and endometrium. These results demonstrate the inhibition of mitotic activity in cervical and endometrial cells brought on by a copper IUD, especially during the 1st 2 months. This action could adversely affect a fetus if pregnancy occurs at this time. The researchers suggest that health practitioners advise any pregnant woman who had only recently got a copper IUD to abort the fetus.^ieng

Effect of phenytoin on mitotic activity of gingival tissue and cultured fibroblasts.

The effect of phenytoin on the mitotic activity of gingival tissue as well as cultured mammalian fibroblasts was studied. This was measured quantitatively using autoradiography utilizing tritiated thymidine. Hyperplastic gingiva removed from patients treated with phenytoin and subjected to autoradiography showed a marked increase in the mitotic activity of gingival cells. This was also the case with cultured fibroblasts. The mitotic index was higher in the treated fibroblasts than that of the control. Ascorbic acid added to the cultures markedly reduced the mitotic activity of these cells. These results agree with other studies that phenytoin may be a factor in direct stimulation of the mitotic activity and gingival hyperplasia seen in patients taking this medication.

Congo/Crimean haemorrhagic fever in Iraq.

Congo/Crimean haemorrhagic fever was recognized for the first time in Iraq in 1979. The first case was reported on 3 September 1979 and since then a further 9 patients have been investigated. Eight patients gave a history of previous contact with sheep or cattle, while 2 patients, a resident doctor and an auxiliary nurse, acquired their infections in hospital by direct contact with patients. The causal virus was isolated from patients' blood and postmortem liver specimens. The virus isolates were found to be closely related if not identical serologically to members of the Congo/Crimean haemorrhagic fever virus group. Eight of the patients had no epidemiological relationship to one another and lived in widely separated areas around Baghdad and Ramadi (110 km to the west of Baghdad).

Crimean-Congo haemorrhagic fever virus in Iraq: isolation, identification and electron microscopy.

Crimean-Congo haemorrhagic fever (CCHF) virus was isolated for the first time in Iraq from the blood of three patients. It caused a cytopathic effect in lamb kidney and BHK-21 cell cultures. The virus particles were spherical, enveloped and had 90 nm in diameter similar particles were found in ultrathin sections of the liver from two fatal cases. The isolated virus proved to be antigenically closely related to CCHF virus.

Elasticity of frozen aortic valve homografts.

Homograft aortic valves were treated and stored in different ways and then tested for elastic qualities. It appears from the findings that the most satisfactory long-term storage technique is to flash-freeze the valves in the presence of dimethyl sulphoxide immediately after dissection.

Viability and histopathology of eleven fresh, antibiotic-treated allografts removed three weeks to three years after implantation.

Changes in metabolic activity of removed heart valve allografts have been measured. The fresh heart valves were sterilized and stored in antibiotic solution before implantation in patients. Viability was determined before insertion and after removal from patients by two methods: 1) tissue culture, and 2) autoradiography, using tritiated thymidine. The length of storage in the Hank's antibiotic or nutrient-antibiotic medium before insertion did not seem to influence the final metabolic activity nor the structural integrity of the allografts when they were removed. Results from the present study show that the most severe degenerative changes occur in valves stored in Hank's solution and then implanted in the mitral position. The viability percentage declined progressively during the time that a valve treated in this manner was functioning in a patient.

Fascia lata valves: a clinicopathological study.

Sixteen frame-mounted fascia lata valves removed from the mitral, aortic or--in one patient--pulmonary position have been detailed histologically. These valves had remained in 15 patients (11 men and four women) for periods varying between 10 and 44 months. The reason for the original transplantation was either chronic rheumatic endocarditis or calcific aortic disease. In the mitral position, the leaflet in position nearest the site of the original anterior mitral valve cusp showed the least changes. The remaining two leaflets of the fascia lata valve in the mitral position, as well as those removed from the aortic or pulmonary position, showed more severe changes; these consisted of degeneration of collagen tissue and often a severe decrease of nuclei belonging to the fibroblastic series. These changes, as well as superimposition of fibrin or fibrous tissue, tended to become more pronounced the longer the valve had remained in the patient. Viability studies in valves removed from two patients have also been undertaken showing very greatly reduced activity. The possible causes for valve dysfunction have been reviewed, and the findings in this study suggest that contraction of fibrous tissue, which sandwiches the fascia lata valve cusps, may contribute to failure of satisfactory valve function. It is concluded that fascia lata forms a poor substitute for replacement of diseased cardiac valves.

Analysis of homograft valve failure in 311 patients followed up to 10 years.

Homograft aortic valve replacement was performed in 311 patients at the tnational Heart thospital, London, between 1964 and 1973. Valve failure has occurred in 61 patients (20%), 32 of whom survived reoperation. From 1963 through 1967, 156 valves were freeze-dried and account for 56 of the valve failures. From 1968 to 1973, 118 fresh or fresh-frozen valves resulted in only 5 failures. Six general types of failure have been identified: calcification (13), dehiscence (15), infective endocarditis (17), prolapse (6), cusp degeneration (5), and tear or perforation (5). Valve failure may be due to surgical technical error resulting in dehiscence or valve incompetence, or it may be related to degenerative changes in the homograft. The clinical results, supported by gross and histological examination and viability testing, enable us to conclude that fresh or fresh-frozen valves are superior to freeze-dried valves, having resulted in only 4% valve failure over the past five years.