Anne-Laure Millard

Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock‐out pigs

Puga Yung GL, Li Y, Borsig L, Millard A‐L, Karpova MB, Zhou D, Seebach JD. Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock‐out pigs. Xenotransplantation 2012; 19: 196–206.

Brief Exercise Increases Peripheral Blood NK Cell Counts without Immediate Functional Changes, but Impairs their Responses to ex vivo Stimulation

Physical as well as psychological stress increases the number of circulating peripheral blood NK cells. Whereas some studies found a positive correlation between exercise and NK cell counts and cytotoxic activity, others showed that, for example, heavy training leads to a decrease in per cell NK cytotoxicity. Thus, the impact of exercise on NK cell function and eventually on altered immunocompetence remains to be elucidated. Here, we investigated whether a single bout of brief exercise, consisting in running up and down 150 stair-steps, affects the number and function of circulating NK cells. NK cells, obtained from 29 healthy donors, before and immediately after brief exercise, were assessed for numbers, phenotype, IFNγ production, degranulation, cytotoxicity, and in vitro response to stimulation with IL-2, IL-2/IL-12, or TLR2 agonists. Running resulted in a sixfold increase in the number of CD3−/CD56+ NK cells, but decreased the frequency of CD56bright NK cells about twofold. Brief exercise did not significantly interfere with baseline IFNγ secretion or NK cell cytotoxicity. In vitro stimulation with IL-2 and TLR2 agonists (lipoteichoic acid, and synthetic triacylated lipopeptide Pam3CSK4) enhanced IFNγ-secretion, degranulation, and cytotoxicity mediated by NK cells isolated pre-exercise, but had less effect on NK cells isolated following exercise. There were no differences in response to combined IL-2/IL-12 stimulation. In conclusion, having no obvious impact on baseline NK functions, brief exercise might be used as a simple method to significantly increase the number of CD56dim NK cell available for in vitro experiments. Nevertheless, the observed impaired responses to stimulation suggest an alteration of NK cell-mediated immunity by brief exercise which is at least in part explained by a concomitant decrease of the circulating CD56bright NK cell fraction.

Efficiency of porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin

Millard A‐L, Häberli L, Sinzger C, Ghielmetti M, Schneider MKJ, Bossart W, Seebach JD, Mueller NJ. Efficiency of porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin.
Xenotransplantation 2010; 17: 274–287.

Human CMV infection of porcine endothelial cells increases adhesion receptor expression and human leukocyte recruitment.

Background. Potential xenozoonosis is a concern for the clinical application of xenotransplantation. Human cytomegalovirus (HCMV) is one of the most important pathogens in allotransplantation, but the consequences of HCMV cross-species infection of porcine xenografts are unknown. Therefore, we investigated the effects of HCMV infection of porcine endothelial cells (pEC) on cell surface molecule expression and human leukocyte recruitment. Methods. Infection of pEC inoculated with untreated, UV-inactivated, or heparin-treated HCMV at a multiplicity of infection (MOI) of 1 was analyzed by immediate early (IE) antigen expression. Cell surface receptor expression was studied by flow cytometry on pEC bulk cultures and differentially on IE-positive and -negative pEC. Adhesion of human leukocytes was tested on pEC monolayers. pEC supernatants were analyzed for cytokine content, chemotactic activity, and stimulatory effect on resting secondary pEC cultures. Results. At day 2 postinfection, IE staining was evident in 10% to 20% of HCMV-infected cells. Cell-surface expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) was upregulated in both IE-negative and -positive fractions of HCMV-infected pEC. In contrast, porcine major histocompatibility complex class I expression was upregulated in IE-negative cells, but reduced in IE-positive cells. The receptor alterations in the IE-negative fraction were mediated by pEC-derived soluble factors. The increased adhesion receptor expression was paralleled by enhanced human leukocyte chemotaxis and adhesion to infected pEC cultures. Pretreatment of HCMV with heparin, but not UV-inactivation, prevented adhesion-receptor modulation and reversed the increased adhesion and chemotaxis. Conclusions. After pig-to-human solid organ transplantation HCMV may infect and activate the porcine endothelium, rendering the xenograft more susceptible to human leukocyte recruitment and rejection.

Inhibition of direct and indirect TLR-mediated activation of human NK cells by low molecular weight dextran sulfate.

NK cells express toll-like receptors (TLR) that recognize conserved pathogen or damage associated molecular patterns and play a fundamental role in innate immunity. Low molecular weight dextran sulfate (DXS), known to inhibit the complement system, has recently been reported by us to inhibit TLR4-induced maturation of human monocyte-derived dendritic cells (MoDC). In this study, we investigated the capability of DXS to interfere with human NK cell activation triggered directly by TLR2 agonists or indirectly by supernatants of TLR4-activated MoDC. Both TLR2 agonists and supernatants of TLR4-activated MoDC activated NK cells phenotypically, as demonstrated by the analysis of NK cell activation markers (CD56, CD25, CD69, NKp30, NKp44, NKp46, DNAM-1 and NKG2D), and functionally as shown by increased NK cell degranulation (CD107a surface expression) and IFN-gamma secretion. DXS prevented the up-regulation of NK cell activation markers triggered by TLR2 ligands or supernatants of TLR4-activated MoDC and dose-dependently abrogated NK cell degranulation and IFN-gamma secretion. In summary our results suggest that DXS may be a useful reagent to inhibit the direct and indirect TLR-mediated activation of NK cells.

Statins demonstrate a broad anti-cytomegalovirus activity in vitro in ganciclovir-susceptible and resistant strains

Vasculoprotective and cholesterol‐lowering properties are hallmarks of statins. Recently, statins have been found to exhibit antiviral activity. Little is known about the potential of statins against human cytomegalovirus (HCMV), a risk factor in the pathogenesis of atherosclerosis. In this study, the in vitro anti‐CMV activity of four statins (atorva‐, fluva‐, prava‐, and simvastatin) was explored in human aortic endothelial cells (HAEC) and fibroblasts. All statins dose‐dependently reduced HCMV titers in both cell types. Whereas atorva‐, fluva‐, and simvastatin showed comparable EC50 and EC90 within a low micromolar range in HAEC, pravastatin exhibited only limited effects. In metabolite rescue experiments, mevalonate almost completely abrogated the anti‐CMV activity of all statins, whereas cholesterol failed to counteract the effects. Geranylgeranyl‐pyrophosphate partially reversed the anti‐CMV activity of most statins, suggesting an involvement of the non‐sterol isoprenoid arm of the mevalonate pathway as the mode‐of‐action. The accumulation of immediate early viral antigens was blocked after 1 dpi onwards, and early and late antigen expression was completely abolished in HAEC. The antiviral activity of statins was comparable to ganciclovir and was retained in a ganciclovir‐resistant HCMV strain. These findings provide new insight into the beneficial effects of statins, adding antiviral activity against HCMV to their list of pleïotropic properties, and support further clinical investigations on combined therapy for the management of active HCMV disease. J. Med. Virol. 87: 141–153, 2015.

Entry of human cytomegalovirus into porcine endothelial cells depends on both the cellular vascular origin and the viral strain.

Primary infection and reactivation of human cytomegalovirus (HCMV) is associated with allograft rejection. Pig‐to‐human xenotransplantation is regarded as an alternative to circumvent donor organ shortage and inevitably, porcine endothelial cells (pEC) will be exposed to human pathogens, among them HCMV. Infection of pEC with HCMV induces apoptosis and entry is sufficient to induce phenotypic alterations, which have the potential to result in rejection and vasculopathy. We investigated the mechanisms used by HCMV to enter pEC from different anatomical origins and compared them with the entry mechanisms used to enter human endothelial cells (hEC).

Critical issues related to porcine xenograft exposure to human viruses: lessons from allotransplantation.

Purpose of reviewXenotransplantation of tissues from swine into humans poses the threat of bidirectional transfer of porcine or human microorganisms to the recipient or to the xenograft, respectively. This review focuses on recipient-derived infection. Recent data are reviewed that assess the susceptibility of porcine cells to human viruses. On the basis of the experience in allotransplantation, potential consequences for the xenograft are discussed. Recent findingsTraditionally, research on xenoses has focused on donor, that is pig-derived, infections. Efforts to exclude pathogens from pig donors have been successful with notable exceptions such as the genetically encoded porcine endogenous retrovirus. Intrinsic resistance of many viruses to infect cells from a different species has been assumed and may confer an advantage for a xenograft. Recent studies, however, have demonstrated the ability of a number of human viruses relevant in allotransplantation to infect porcine cells. Infection was associated with cytopathogenicity as well as cellular changes promoting adhesion and transmigration of human cells or a procoagulant stage. SummarySuccessful infection of porcine cells with human viruses has challenged the concept of species specificity. For some viruses, infection resulted in production of infective progenies and is associated with cytopathogenicity. Cellular alterations potentially enhance the risk for graft damage, rejection or coagulation abnormalities.