US28 actions in HCMV infection: lessons from a versatile hijacker.

Mimicking host proteins is a strategy adopted by several herpesviruses to exploit the host cell for their own benefit. In this respect the human cytomegalovirus (HCMV) chemokine receptor homologue US28, has been extensively studied. Molecular pirates such as US28 can teach us about crucial events in HCMV infection and may either offer a potential target for antiviral therapy or provide an alternative strategy to immune suppression. Despite elaborate research into the chemokine binding affinity, signalling properties, intracellular trafficking and expression kinetics of US28, a solid hypothesis about the role of US28 in HCMV infection has not yet been proposed. It appears that US28 may behave as a molecular pirate that employs smart strategies for cell entry, host gene regulation and immune evasion. This review will elaborate on these aspects of US28 biology and discuss possible implications for HCMV infection.

The value of prospective monitoring of Epstein-Barr virus DNA in blood samples of pediatric liver transplant recipients.

UNLABELLED: Post-transplant lymphoproliferative disease (PTLD) is one of the major causes of morbidity and mortality in transplantation patients. A primary Epstein-Barr virus (EBV) infection is a major risk factor for developing PTLD. The aim of this study was to determine circulating EBV DNA after liver transplantation in pediatric patients in relation to primary EBV infection and development of PTLD. EBV serology was performed before transplantation. Every 4 weeks after transplantation a competitive quantitative polymerase chain reaction (PCR) assay for EBV nuclear antigen-1 was performed in 13 patients. Patients were followed for development of a PTLD. Before transplantation four patients were EBV seropositive and nine patients were EBV seronegative. In one of the four patients who were EBV seropositive before transplantation, EBV DNA became detectable after transplantation, with a peak load of 3600 copies/mL. None of these four patients developed a PTLD. Eight of the nine patients who were EBV seronegative before transplantation developed positive EBV DNA samples. EBV DNA was first detected at a mean of 64 days after transplantation (range 38-89). The mean peak EBV DNA load was 79,700 copies/mL (3600-446,000). Two of these patients developed PTLD, but they could not be identified based on prior or concomitant EBV PCR results. CONCLUSIONS: In pediatric liver transplantation EBV DNA load is higher in patients with a primary infection than in patients who were EBV seropositive before transplantation. The EBV PCR cannot be used to identify individual patients who develop PTLD. However, elevated EBV DNA load can be used to detect a group of patients at increased risk for PTLD.

Pulmonary involvement during cytomegalovirus infection in immunosuppressed patients.

Although cytomegalovirus (CMV) pulmonary involvement after solid organ transplantation is infrequently seen nowadays, CMV pneumonitis is still a potential lethal complication. Introduction of the pp65 antigenemia assay enabled early and rapid diagnosis of CMV viremia in transplant patients prior to symptoms. Also, in asymptomatic patients with CMV viremia, a decreased pulmonary diffusion capacity could be demonstrated. In this review, we discuss clinical and subclinical pulmonary involvement of CMV infection in the immunocompromised host with an emphasis on transplant recipients. The clinical course, diagnosis, therapy, prophylaxis, and pathophysiology of CMV pneumonitis are discussed.

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo.

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.

Dendritic cells present an intracellular viral antigen derived from apoptotic cells and induce a T-cell response.

We investigated if dendritic cells (DCs) were able to present intracellularly located antigens derived from apoptotic cells to T cells, thereby inducing a CD4+ and a CD8+ response. A transfected cell line with the cytomegalovirus-derived protein pp65 was triggered to go into apoptosis by ultraviolet B (UVB) irradiation, and after the uptake of apoptotic cells by DC, the activation and proliferation of T cells were determined. We found that DC efficiently phagocytosed apoptotic cells and induced a CD4+ and a CD8+ T-cell response specific for the viral protein pp65. This mechanism can be useful for vaccination studies to induce an antiviral immune response.

Effects of changing immunosuppressive regimen on the incidence, duration, and viral load of cytomegalovirus infection in renal transplantation: a single center report.

Background. In this retrospective single center study we have evaluated the relation between the immunosuppressive regimen and the incidence and characteristics of cytomegalovirus (CMV) infection in the setting without CMV prophylaxis from 1989 through 1998. Methods. All (470) first cadaveric renal transplantations in nonsensitized (PRA < 60%) patients were analyzed. Immunosuppression consisted of cyclosporine A (Sandimmune) and prednisolone from 1989 through 2-1993 (S; 189 patients), of cyclosporine microemulsion (Neoral) and prednisolone from 3-1993 through 5-1997 (N; 200 patients) and of mycophenolate mofetil, Neoral and prednisolone from 5-1997 until 1998 (M; 81 patients). The CMV pp65-antigenemia was measured routinely at least once weekly from day 10 till 12 weeks after transplantation or until pp65-antigenemia became negative. No CMV-prophylaxis was given. Results. By changing from Sandimmune to Neoral and by adding mycophenolate mofetil, respectively, we observed a higher frequency of especially secondary CMV infections (S vs. N vs. M, respectively, 28 vs. 50 vs. 63%, P = 0.026; S vs. N, P = 0.027; S vs. M, P = 0.015; and N vs. M, n.s). The CMV infections lasted longer (median duration antigenemia S vs. N vs. M, respectively, 3 vs. 5 vs. 7 weeks, P = 0.0003; S vs. N, P < 0.002; S vs. M, P < 0.001; and N vs. M, P < 0.05). Viral load was higher in M (median maximal pp65-antigenemia S vs. N vs. M, respectively, 19 vs. 14.5 vs. 73, P < 0.01; S vs. N, n.s.; S vs. M, P < 0.001 and N vs. M, P < 0.01). Conclusions. The use of Neoral and the addition of mycophenolate mofetil caused significant changes in the incidence, duration and viral load of CMV infections.

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA.

Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for the removal of proteins without histidine tag from IMAC columns. Four histidine-tagged recombinant proteins/protein complexes were purified to homogeneity from cell culture medium of insect cells by using an EDTA washing buffer. The presence of a low concentration of EDTA in washing buffers during IMAC may have a general application in the purification of histidine-tagged proteins.

Expression dynamics of human cytomegalovirus immune evasion genes US3, US6, and US11 in the blood of lung transplant recipients.

Delayed elimination of human cytomegalovirus (HCMV)-infected cells by the host immune system may contribute to viral dissemination and pathogenesis of HCMV infection. The mRNA expression dynamics of HCMV-encoded immune evasion genes US3, US6, and US11 expressed after active HCMV infection were analyzed in blood samples of lung transplant recipients by means of quantitative nucleic acid sequence-based amplification. The results were compared with the expression dynamics of IE1 mRNA and pp67 late mRNA, levels of pp65 antigenemia, and antiviral treatment. During acute infection, high levels of US3 and US6 RNA were detected before antigenemia, which were detected simultaneously with IE1 RNA. US11 RNA was detected simultaneously with antigenemia but before late pp67 RNA. These data suggest an active role of viral immune evasion during HCMV infection in vivo. Interestingly, immune evasion RNA remained detectable after clinical recovery, often independently of IE1 RNA expression, indicating persistent viral activity, which may have implications for long-term control of HCMV.

Susceptibility to development of Mycobacterium ulcerans disease: review of possible risk factors.

Mycobacterium ulcerans disease, also known as Buruli ulcer (BU), is a disease of subcutaneous fat tissue. BU is prevalent in riverine and swamp areas of the tropical zone in Africa, Asia and South America, and a few scattered foci in Australia. The mode of transmission of M. ulcerans has not been fully elucidated, but inoculation into the subcutaneous tissues probably occurs through penetrating skin trauma. BU has not been linked with HIV infection. Antimycobacterial drug treatment is ineffective, and treatment is surgical. Patients eventually develop scars and contractures, with resulting disabilities, and the disease imposes a large burden on affected populations. The incidence of BU has dramatically increased in West African countries over the last decade. There is an urgent need for research into host and environmental risk factors for BU in order to develop effective strategies to combat this disease. We review possible genetic host susceptibility factors for BU that are relevant in other mycobacterial diseases: natural resistance-associated macrophage protein-1 (NRAMP-1), HLA-DR, vitamin D3 receptor, mannose binding protein, interferon-gamma (IFN-gamma) receptor, tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1 alpha, 1 beta and their receptor antagonists; and IL-12. Schistosoma haematobium infection is highly endemic in many BU foci in West Africa, with a striking increase in transmission after river dams were constructed. This observation, and the observations from interaction of schistosomiasis and tuberculosis, have fueled our hypothesis that schistosomiasis is a risk factor for BU by driving the host immune response towards a predominantly Th-2 pattern, away from a Th-1 preponderant protection against mycobacterial infection. If the latter hypothesis is confirmed, enhanced schistosomiasis control should impact on BU.

Viral load in breast milk correlates with transmission of human cytomegalovirus to preterm neonates, but lactoferrin concentrations do not.

In vitro, lactoferrin (LF) strongly inhibits human cytomegalovirus (HCMV), which led us to hypothesize that in vivo HCMV might also be inhibited in secretions with high LF concentrations. In breast milk, high viral loads observed as high viral DNA titers tended to coincide with higher LF levels. However, the LF levels did not correlate to virus transmission to preterm infants. The viral load in the transmitting group was highest compared to the nontransmitting group. We conclude that viral load in breast milk is an important factor for transmission of the virus.

Uptake of pp65 in in vitro generated pp65-positive polymorphonuclear cells mediated by phagocytosis and cell fusion?

OBJECTIVE: The cytomegalovirus (CMV) antigenemia consists of the detection of CMV pp65 in the nucleus of polymorphonuclear granulocytes (PMN), but it is unclear where and how PMN pick up virus particles or proteins. In an in vitro model for CMV antigenemia we investigated the mechanism of pp65 uptake by PMN that results in its expression in the nucleus. METHODS: A series of inhibitors of different mechanisms was used to study the uptake of pp65 by PMN during coculture with CMV-infected endothelial cells and we performed a morphological analysis by light and transmission electron microscopy. RESULTS: Nocodazole and cytochalasin B inhibited uptake of pp65 by PMN with 59.4 +/- 14.1 and 73.3 +/- 12.7%, respectively. The presence of anti-CMV hyperimmune globulin or lactoferrin during coculture reduced the number of pp65-positive PMN with 45.8 +/- 7.0 or 40.6 +/- 3.2%. Furthermore, a small number of the pp65-positive PMN obtained after coculture had fused to large cells with multilobed nuclei. PMN were observed that enclosed viral particles as well as free viral particles containing PMN in the cytoplasm. CONCLUSION: Fusion of viral particles with PMN and phagocytosis are both involved in the uptake of pp65.

Cytomegalovirus infection increases the expression and activity of ecto-ATPase (CD39) and ecto-5'nucleotidase (CD73) on endothelial cells.

We describe enhanced expression and enzymatic activity of ecto-ATPase and ecto-5'nucleotidase on CMV infected endothelial cells as compared to uninfected cells. These ectoenzymes play a major role in modulation of platelet activation and aggregation. Furthermore, adenosine has a modulatory effect upon inflammation. Addition of ATP, ADP or AMP to cultures of CMV infected or uninfected endothelial cells revealed increased turnover of AMP in CMV infected endothelial cells. In addition, the superoxide production by stimulated polymorphonuclear cells was inhibited in the presence of CMV infected endothelial cells as compared to uninfected cells, probably due to the enhanced activity of ecto-5'nucleotidase and associated to production of adenosine.

Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients.

Posttransplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplantation recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per mL blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3.4% of the whole blood samples was above the cut-off value (P <.0001) despite heavy immune suppression and cytomegalovirus (CMV)-related disease. These findings illustrate the clinical importance of frequent EBV DNA load monitoring in LTx recipients. The increased EBV DNA loads in PTLD patients were restricted to the cellular blood compartment, as parallel serum samples were all below cut-off value, which indicates absence of lytic viral replication. EBV(+) cells in PTLD patients have a very short doubling time, which can be as low as 56 hours, thereby creating the need for high screening frequency in high-risk patients. Furthermore, it is shown that EBV and CMV can reactivate independently in LTx recipients and that EBV DNA load monitoring may be useful in discriminating PTLD from rejection.

Direct quantification of human cytomegalovirus immediate-early and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification.

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10(4) copies per 100 microl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.

Acute rejection before cytomegalovirus infection enhances von Willebrand factor and soluble VCAM-1 in blood.

BACKGROUND: Human cytomegalovirus (HCMV) infections in transplantation patients are associated with vascular endothelial damage. This is reflected by the appearance of cytomegalic endothelial cells (CECs) and noninfected endothelial cells (ECs) in blood. To get more insight in the extent of vascular damage during HCMV infection, we investigated the levels of soluble markers during HCMV infection in relationship to EC levels and also preceding the acute rejection episodes. METHODS: Of 46 kidney transplant patients, plasma levels of von Willebrand factor (VWF), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble E-selectin (sE-sel) were analyzed during the course of HCMV infection. RESULTS: Plasma levels of VWF and sVCAM-1 increased twofold during severe HCMV infection. Moreover, the plasma levels of VWF correlated with detectable cytomegalic and noninfected ECs in blood. The kinetics of changes in VWF and ECs (CEC and EC) demonstrated the relationship with HCMV-induced vascular damage. Levels of sICAM-1 and sE-sel in plasma did not significantly change during HCMV infection. Interestingly, the combination of HCMV infection and preceding acute transplant rejection caused the highest increases of VWF and sVCAM-1 plasma levels, reflecting an enhanced susceptibility for endothelial damage at the moment of infection. CONCLUSION: CMV infection is associated with vascular damage, and the vascular damage during CMV infection is enhanced if patients experienced acute rejection before CMV infection.