Dendritic cells and HCMV cross-presentation.

The outcome of a viral infection is the result of an endless fight between the organism whose task is to mount an antiviral response and the virus that adapts strategies to circumvent the host response. Human cytomegalovirus (HCMV), a latent herpesvirus, can be considered as a spearhead in exploiting co-existence with the host to develop numerous immuno-evasion mechanisms. The ability of the organism to initiate a primary immune response against viruses such as HCMV is highly dependent on the capacity of professional antigen-presenting cells (APCs), namely dendritic cells (DCs), to prime and activate specific effector T cells. Recent findings emerging from the murine cytomegalovirus (MCMV) animal model demonstrated that infection of murine DCs with MCMV impaired their capacity to prime an effective T cell response. Even though data on interference of HCMV with DC functions are still limited, immunosuppressive effects identical to those reported for MCMV can be suspected and we may then ask how a cytotoxic T lymphocyte (CTL) response is generated in these unfavourable conditions. In response to this question, cross-presentation of HCMV antigens by uninfected DCs to CD8+ T cells could be considered a key process in initiating an immune response. In this chapter we discuss the mechanisms through which DCs could acquire HCMV antigens and how cross-presentation could be modulated throughout infection. Moreover, further knowledge of DC functions is key for the development of DC-based immunotherapy against HCMV.

Differential modulation of complement factor H and C3 expression by TNF-alpha in the rat. In vitro and in vivo studies.

The biosynthesis of alternative regulatory complement protein factor H was investigated using both an in vivo rat model and an in vitro rat hepatocyte culture system, and compared to that of C3 component. Subcutaneous injection of a single dose of 20 micrograms of recombinant murine tumor necrosis factor-alpha (rmTNF-alpha) had no effect on factor H liver mRNA levels, while it increased C3 mRNA levels. In correlation with this, serum factor H levels remained unchanged after rmTNF-alpha injection, whereas C3 levels were increased. In contrast in vitro studies showed that rmTNF-alpha had no effect on factor H and C3 expression by rat hepatocytes. Recombinant human interleukin-1 alpha (rhIL-1 alpha) did not alter the expression of factor H, whereas it increased C3 expression, and recombinant human interleukin-6 (rhIL-6) stimulated expression of both proteins. This study shows that TNF-alpha is not directly responsible for the increased levels of factor H observed in vivo during induced inflammation in the rat. Its in vivo effect on C3 secretion might be secondary to the TNF-alpha-induced release of IL-1 and/or IL-6.

Partial hepatectomy and mediators of inflammation decrease the expression of liver alpha 2-HS glycoprotein gene in rats.

Liver mRNA levels of two acute phase reactant (APR) proteins, alpha 2-HS glycoprotein (a major negative APR) and alpha 1-acid glycoprotein (a major positive APR) were measured in male rats at different times after the administration of turpentine, of tumor necrosis factor, or following partial hepatectomy. In every case, a marked decrease in mRNA levels of alpha 2-HS glycoprotein was observed which reached a maximum at 24 h. A concomitant increase of alpha 1-acid glycoprotein mRNA levels was observed under the same conditions. These results indicate that the decreased levels of alpha 2-HS glycoprotein induced by the acute-phase response following inflammatory mediators and partial hepatectomy are due to a down-regulation of the gene expression of this protein in rat liver.

An improved three-step method for the purification of the third component of human complement.

This paper describes a three-step purification method for the third component of human complement (C3) from plasma. The method consists of PEG precipitation, DEAE-cellulose chromatography and preparative isofocusing in a granulated dextran gel, with pH range 5--8. From this last step, a highly purified form of native C3 was obtained as indicated by immunoelectrophoresis and polyacrylamide gel electrophoresis analysis. The average final recovery was 20%.

Human cytomegalovirus infection induces leukotriene B4 and 5-lipoxygenase expression in human placentae and umbilical vein endothelial cells.

INTRODUCTION: Human cytomegalovirus (HCMV) can cause congenital infection with risk of neurological disability. Maternal-fetal transmission is associated with placental inflammation. 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of Leukotrienes (LTs), which are proinflammatory mediators. This study investigated the effect of HCMV infection on 5-LO expression and Leukotriene-B4 (LTB4) induction in human placentae and umbilical vein endothelial cells (HUVEC). METHODS: Seven placentae from fetuses with congenital HCMV infection and brain damage and six controls were stained with HCMV-immediate-early-antigen (HCMV-IEA) and 5-LO by immunohistochemistry. 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4 were measured in culture supernatant from ex vivo HCMV-infected placental histocultures by liquid chromatography. In vitro, HCMV infected HUVEC cells were analyzed for 5-LO mRNA and protein expression by real time PCR and immunofluorescence staining. RESULTS: HCMV-IEA was abundant in all HCMV infected placentae but absent in control placentae. 5-LO expression was higher in endothelial and smooth muscle cells of HCMV-infected placentae, compared to control placentae. HCMV infection induced an up-regulation of LTB4 in ex vivo placental explants with higher levels of LTB4 at 72 h compared to controls (p = 0.002). In vitro, 5-LO transcript and protein expression were significantly induced in HCMV-infected HUVEC, compared to the control cultures (p = 0.036). CONCLUSION: The presence of HCMV coincided with high 5-LO expression in cells of in vivo HCMV infected placentae. HCMV induced up-regulation of 5-LO in both ex vivo HCMV-infected placental explants and HUVEC. HCMV induced LT-biosynthesis in congenitally infected placentae may have a role in pathogenesis of congenital HCMV disease.

Novel model of placental tissue explants infected by cytomegalovirus reveals different permissiveness in early and term placentae and inhibition of indoleamine 2,3-dioxygenase activity.

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Placental infection suggests hematogenous spread and permissiveness may vary according to the age of pregnancy. We set up and investigate permissivity of early and term placenta to HCMV with an ex vivo model of placental histocultures and evaluate the activity profile of IDO. Fourteen first trimester placentae were obtained following elective abortion and twelve term placentae after elective caesarean section. Fresh placental chorionic villi were isolated, washed and distributed on collagen sponge gels after overnight incubation with the virus. The culture medium was collected and fresh medium renewed regularly. Histology and immunohistochemistry showed preserved villous integrity in cultured placental histocultures. Infection could be seen in tissue sections of both early and term placentae, although early placentae were more permissive. Indoleamine 2,3-dioxygenase (IDO) is highly expressed in the placenta and is known to prevent maternal immune rejection. Constitutive IDO activity was higher in early, compared to term placentae and HCMV infection inhibited IDO activity in early placentae. IFN-γ-induced IDO activity was suppressed by HCMV in both early and term placentae. Our work shows a novel method of placenta organ culture. Our findings suggest that HCMV infects early placentae more strongly than term placentae. Early placental dysfunction through the inhibition of IDO activity may reveal a possible mechanism for miscarriages.

Polymorphism of the third component of human complement.

Genetic polymorphism of the third component of human complement (C3) has been considered as a powerful marker for population genetics. Some studies on the distribution of gene frequencies have been performed among numerous populations all over the world. This review takes stock of population genetic studies reported up to now and points out some remarks on the distribution of the observed allelic frequencies.

BF DNA reference typing.

A study of the RFLP patterns of the samples submitted, using TaqI and MspI restriction enzymes has allowed a confirmation of the correlation previously reported on the BF protein types and subtypes. The 6.6-kb band obtained with TaqI is closely correlated to the BF FA type and the MspI 0.7-kb band with BF F.

Molecular characterization of human complement factor B subtypes.

While several laboratories have agreed that there are two subtypes of the BF*F alleles, no information has been available until now at the molecular level. The region of the BF gene corresponding to the Ba fragment [1.7 kilobases (kb)] of the BF*S, BF*FA, and BF*FB alleles has been sequenced after specific amplification using the polymerase chain reaction (PCR). A point mutation at codon 7 has been revealed converting a cytosine in the BF*S allele to a thymidine in BF*FB. At the translational level an arginine residue in BF*S is substituted for a tryptophan residue in BF*FB. The amino-terminal sequencing of factor B immunoprecipitated from serum has been carried out from microquantities of protein blotted onto polyvinylidene fluoride (PVDF) membranes. We have shown that the difference between the BF*FA and the BF*FB subtypes in characterized by a glutamine at position 7 in BF*FA and a tryptophan in BF*FB.

Prevalence of the 1.8 kb complement factor H mRNA in human lung.

Human complement factor H, described as a 155,000 molecular weight (MW) component, is a key factor in the control of the alternative pathway of complement activation. Using two human factor H cDNA clones, designated R2a (a clone derived from the 3' end of the factor H coding sequence) and B38-1 (a clone derived from the 5' end of the factor H coding sequence), as probes, three factor H-specific transcripts of 4.3 kb, 1.8 kb and 1.5 kb are usually detected in human liver, in equal abundance. Using these two factor H cDNAs to probe human lung RNA, there was evidence of a singular distribution of the factor H mRNA species in human lung compared to liver, in equal prevalence of the 1.8 kb factor H mRNA over the 4.3 kb factor H mRNA (a three- to fourfold difference). No significant expression of the 1.5 kb mRNA was detected. The prevalence of the 1.8 kb complement factor H mRNA leads to the speculation that the predominant factor H form biosynthesized in lung tissue is a truncated form of the factor H molecule.

Heterogeneous nature of human complement factor B: an electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products.

Factor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine-linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro-secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.

Allotypic variants of human C3 and properdin factor B in two French populations Normans and Basques.

Human Properdin factor B and C3 polymorphisms are of a real usefulness as genetic markers for population studies. In addition, they are more and more used for the determination of paternity cases. The present communication will be about Bf and C3 allele frequencies in two locally populations of France, Normandy and Western Pyrenees (Basques), The corresponding allele frequencies are as follows, for Normans : Bf(S) = 0.7422, Bf(F) = 0.2285, Bf(F1) = 0.0205, Bf(SO.7) = 0.0088; CsS = 0.788, C3F = 0.204, C3Srare = 0.004, C3Frare =0.004; and for Basques : BfS = 0.5625, BfF = 0.305, BfF1 = 0.1235, BfSO.7 = 0.0075; C3S = 0.7075 and C3F - 0.2925. French Basques are characterized by unusual allele frequencies in European Caucasoid populations for Bf as for C3 genes. A very high incidence of BfF1 allele is reported (BfF1 = 0.125), as previously observed [7, 17].

Molecular basis for the microheterogeneity of human complement factor B.

The involvement of sialic acids in the microheterogeneity of human complement factor B was investigated. Desialylation kinetics revealed all the charge intermediates from a complex native to a homogeneous form. The relation between this heterogeneity and posttranslational events was explored in cultured hepatoma cells. Intracellular factor B exhibited the same isoelectric focusing pattern as the desialylated purified protein, whereas a highly heterogeneous form was secreted. In contrast, when N-glycosylation was prevented by tunicamycin, both intracellular and secreted forms focused like intracellular factor B from control cultures. These data lead to the conclusion that the microheterogeneity of human factor B results from different degrees of sialylation of its N-glycans.

Genetic variants of factor B in a population of Jordan.

BF phenotyping was performed in a population of Jordan. The observed allele frequencies were as follows: BF*S = 0.5457, BF*F = 0.3744, BF*SO7 = 0.0763, BF*F1 = 0.0075. These values are in agreement with the geographic position and the ethnic composition of Jordan.

Alpha-1-antitrypsin phenotypes in a population of Jordan.

Frequencies of the three common subtypes of PI M were studied in a Jordanian population. In comparison with other populations, PI*M3 was found to be low (0.038) and PI*M2 rather high (0.155).

High-performance liquid chromatographic assay for 3'-amino-3'-deoxythymidine in plasma, with 9-fluorenyl methyl chloroformate as the derivatization agent.

We have developed a sensitive high-performance liquid chromatographic assay for the determination of the zidovudine metabolite 3'-amino-3'-deoxythimidine (AMT) using fluorescence and sensitivity in the picomolar range. Plasma was diluted with 0.05 M sodium phosphate buffer pH 7.2 and subsequently prepared for analysis using solid-phase extraction. AMT was derivatized with 9-fluorenyl methylchloroformate and chromatographed using a reversed-phase system. The mobile phase consisted of acetonitrile-0.01 M potassium phosphate buffer (pH 7) (32.68, v/v). The fluorescence of the column effluent was monitored at 262 nm (excitation) and 306 nm (emission). Good resolution of AMT from endogenous plasma components was obtained. Within- and between-day variability was less than 10%. The limit of quantitation was 0.9 microgram/l. The assay was successfully applied to the determination of AMT in human plasma and in plasma of mice treated with zidovudine.

Distinct pathways for tumor necrosis factor alpha and ceramides in human cytomegalovirus infection.

Human cytomegalovirus (HCMV) infection can be fatal to immunocompromised individuals. We have previously reported that gamma interferon and tumor necrosis factor alpha (TNF-alpha) synergistically inhibit HCMV replication in vitro. Ceramides have been described as second messengers induced by TNF-alpha. To investigate the mechanisms involved in the inhibition of HCMV by TNF-alpha, in the present study we have analyzed ceramide production by U373 MG astrocytoma cells and the effects of TNF-alpha versus ceramides on HCMV replication. Our results show that U373 MG cells did not produce ceramides upon incubation with TNF-alpha. Moreover, long-chain ceramides induced by treatment with exogenous bacterial sphingomyelinase inhibited HCMV replication in synergy with TNF-alpha. Surprisingly, short-chain permeant C6-ceramide increased viral replication. Our results show that the anti-HCMV activity of TNF-alpha is independent of ceramides. In addition, our results suggest that TNF-alpha and endogenous long-chain ceramides use separate pathways of cell signalling to inhibit HCMV replication, while permeant C6-ceramide appears to activate a third pathway leading to an opposite effect.

Inulin-induced activation of factor B in whole serum: description of structural modifications in the Ba fragment.

The investigation of inulin-induced conversion of human factor B in serum by isoelectrofocusing revealed physiological modifications in the primary structure of the Ba fragment. Evidence has been obtained that a nascent Ba, generated by the hydrolytic action of the D component on B in serum, was a short-lived product and that a fast release of carboxy-terminal arginine and lysine residues occurred involving a serum carboxypeptidase B enzyme.

Anti-human cytomegalovirus activity of cytokines produced by CD4+ T-cell clones specifically activated by IE1 peptides in vitro.

The control of latent cytomegalovirus (CMV) infections by the immune system is poorly understood. We have previously shown that CD4+ T cells specific for the human CMV major regulatory protein IE1 are frequent in latently infected healthy blood donors. In order to learn about the possible role of these cells, we have developed IE1-specific CD4+ T-cell clones and, in this study, analyzed their epitope specificity and function in vitro. We measured their cytokine production when stimulated with specific IE1 peptides or whole recombinant IE1 protein. Their cytokine profiles, as deduced from gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) and IL-6 production, were of the Th0- and Th1-like phenotypes. Supernatants from IE1-specific clones producing IFN-gamma and TNF-alpha were shown to inhibit CMV replication in U373 MG cells. This effect was due, as found by using cytokine-specific neutralizing antibodies, mostly to IFN-gamma, which was secreted at higher levels than TNF-alpha. To better assess the anti-CMV activity of cytokines, recombinant IFN-gamma and TNF-alpha were used and shown to have a synergistic effect on the inhibition of CMV replication and protein expression. Thus, IE1-specific CD4+ T cells display in vitro anti-CMV activity through cytokine secretion and may play a role in the control of in vivo latent infections.

C3, BF and C4 polymorphisms in familial Mediterranean fever.

BF, C3 and C4 phenotyping were investigated in 34 patients with familial Mediterranean fever (FMF) and in 48 control subjects. Both groups included Sephardic Jews born in Tunisia, Algeria and Morocco. No linkage between BF, C3 and C4 polymorphisms and FMF was found.