Kinetics of US28 gene expression during active human cytomegalovirus infection in lung-transplant recipients.

Targeting viral proteins early during infection may limit exacerbation of human cytomegalovirus infection. The viral chemokine-receptor homologue US28 interferes with leukocyte trafficking and, possibly, viral replication. Because US28 molecules are abundant on the surface of infected cells, this homologue is a potential target for antiviral therapy. To assess the relationship between US28 and disease activity, we measured, by quantitative reverse-transcription polymerase chain reaction, the levels of US28 and immediate-early (IE) 1 gene transcripts in the blood of lung-transplant recipients. We found that, during primary and secondary infection, the IE1 and US28 genes have early transcription kinetics and are expressed at similar levels. This may render US28 an attractive target for antiviral therapy.

The human cytomegalovirus-encoded receptor US28 increases the activity of the major immediate-early promoter/enhancer.

The activation of the major immediate-early promoter (MIEP) is a key event in the cytomegalovirus replication cycle and is dependent on cellular transcription factors which are partially activated by viral proteins. Expression of the viral chemokine receptor homolog US28 results in constitutive activation of pro-inflammatory transcription factors that may be involved in the activation of the major immediate-early promoter/enhancer. Using reporter gene assays in human embryonic kidney cells, we found that US28 signaling was responsible for increased major immediate-early promoter/enhancer activity which was independent of beta-chemokine binding. Inhibition of nuclear factor-kappaB (NF-kappaB) only partially blocked the effect of US28, whereas treatment with a specific p38 mitogen activated kinase (MAPK) inhibitor fully abrogated the US28-induced enhancement of promoter activity. Our results suggest that during human cytomegalovirus (HCMV) infection, US28 in epithelial cells transactivates the major immediate-early promoter/enhancer via the activation of p38 MAPK and downstream signaling that partially involves NF-kappaB.

Abdominal obesity and smoking are important determinants of C-reactive protein in renal transplant recipients.

BACKGROUND: C-reactive protein (CRP) is a predictor of coronary heart disease, total mortality and chronic allograft nephropathy in renal transplant recipients. The determinants of CRP have been investigated in the general population, but not in renal transplant recipients. CRP might reflect metabolic aberrations in association with central obesity and systemic atherosclerosis. However, it may also reflect a low-grade immune-mediated response to the graft. In this study we investigated the factors associated with CRP in a renal transplant population. METHODS: Between August 2001 and July 2003, renal transplant recipients with a functioning graft for more than 1 year (n = 847) were eligible for investigation at their next visit to the outpatient clinic. A total of 606 patients (55% male, aged 51+/-12 years) participated at a median (interquartile range) time of 6.0 (2.6-11.4) years post-transplant. RESULTS: Median CRP concentration was 2.0 (0.80-4.8) mg/l and mean 24 h creatinine clearance was 62+/-22 ml/min. CRP was significantly associated with body mass index, waist circumference and waist-to-hip ratio (P-value < 0.0001). None of the transplant characteristics except creatinine clearance was associated with CRP. In multiple regression analysis, waist circumference, log sICAM-1 concentration, gender, creatinine clearance and current smoking were independently associated with CRP. CONCLUSIONS: In renal transplant recipients waist circumference and smoking are the two most important modifiable independent determinants of CRP. Furthermore, CRP is independently associated with the endothelial function parameter sICAM-1 and, in univariate analyses, associated with multiple cardiovascular risk factors. CRP is not associated with any of the transplant-related factors, except for renal transplant function.

The modulation of angiogenesis in the foreign body response by the poxviral protein M-T7.

The foreign body response is characterized by enhanced recruitment of inflammatory cells. As the directional movement of cells is controlled by chemokines, disruption of the chemokine network would be an attractive approach to improve biocompatibility of an implanted material. The sequestration of chemokines by cell surface-expressed glycosaminoglycans (GAGs) is vital for in vivo chemokine activity. The myxoma virus encodes a soluble protein, M-T7, that interacts with conserved GAG-binding domains of chemokines to block chemokine-mediated leukocyte recruitment. We hypothesized that M-T7 might also affect the function of other inflammation-associated proteins in addition to chemokines that bind to GAG. In our studies, we focussed on the modulation of the GAG-binding molecules macrophage chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor-164 (VEGF164) in the inflammatory reaction against subcutaneously implanted degradable cross-linked dermal sheep collagen discs in AO rats. Genetic delivery of M-T7 delays the influx of macrophages into the collagen discs. In addition, angiogenesis around the implanted material was reduced. The discs revealed reduced levels of rat MCP-1 and rat VEGF164. This was not due to down regulation of transcription of the genes that encode MCP-1 and VEGF164. Our in vivo observations suggest that, in addition to chemokines such as MCP-1, M-T7 neutralizes VEGF164.

Viral chemokine-modulatory proteins: tools and targets.

The chemokine network is an extensive system that regulates many immune functions such as leukocyte locomotion, T cell differentiation, angiogenesis and mast cell degranulation. Tight control of chemokines is vital for proper immune function. Not surprisingly, viruses have found ways to subvert or exploit the immune system in order to persist in co-existence with their hosts. Several viral immune evasion genes encode proteins that modulate the chemokine network. We attempt to identify which aspects of the chemokine control mechanisms are susceptible to modulation. Chemokine-glycosaminoglycan interaction, extracellular processing of chemokines and chemokine scavenging will be discussed in the light of poxvirus and herpesvirus immune evasion. Viral chemokine-modulatory proteins may either be targets for anti-viral therapy or lead the way to new anti-inflammatory chemokine-modulating drugs.

Insulin resistance as putative cause of chronic renal transplant dysfunction.

Transplantation is the preferred organ replacement therapy for most patients with end-stage renal disease. Despite impressive improvements over recent years in the treatment of acute rejection, approximately half of all grafts will loose function within 10 years after transplantation. Chronic renal transplant dysfunction, also known as transplant atherosclerosis, is a leading cause of late allograft loss. To date, no specific treatment for chronic renal transplant dysfunction is available. Although its precise pathophysiology remains unknown, it is believed that it involves a multifactorial process of alloantigen-dependent and alloantigen-independent risk factors. Obesity, posttransplant diabetes mellitus, dyslipidemia, hypertension, and proteinuria have all been identified as alloantigen-independent risk factors. Notably, these recipient-related risk factors are well-known risk factors for cardiovascular disease, which cluster within the insulin resistance syndrome in the general population. Insulin resistance is considered the central pathophysiologic feature of this syndrome. It is therefore tempting to speculate that it is insulin resistance that underlies the recipient-related risk factors for chronic renal transplant dysfunction. Recognition of insulin resistance as a central feature underlying many, if not all, recipient-related risk factors would not only improve our understanding of the pathophysiology of chronic renal transplant dysfunction, but also stimulate development of new treatment and prevention strategies.

Quantitative Epstein-Barr virus (EBV) serology in lung transplant recipients with primary EBV infection and/or post-transplant lymphoproliferative disease.

The Epstein-Barr virus (EBV)-specific antibody response was studied in lung transplant patients to assess their value in the diagnosis and prognosis of post-transplant lymphoproliferative disease. Recently developed synthetic peptides representing Epstein-Barr nuclear antigen-1 (EBNA-1), diffuse early antigen (EA(D)), and virus capsid antigen (VCA) were studied in a semiquantitative enzyme-linked immunosorbent assay (ELISA) to study antibody patterns in 12 seronegative lung transplant patients, of whom four developed a post-transplant lymphoproliferative disease, and seven seropositive lung transplant patients, all of whom developed a post-transplant lymphoproliferative disease. Immunoblot technique was used as a control. All 12 EBV-seronegative patients had a very limited antibody response that was restricted mainly to VCA antibodies. EA(D) antibodies became detectable in only two patients. Antibody response never preceded clinical diagnosis of post-transplant lymphoproliferative disease in the four EBV-seronegative patients who developed post-transplant lymphoproliferative disease. In the seven seropositive lung transplant patients with post-transplant lymphoproliferative disease, we found a rise in antibody titer in only two patients. Immunoblot analysis confirmed the serological results. In conclusion, EBV-specific antibody patterns after lung transplantation are highly restricted and variable and of limited value for the diagnosis or prognosis of post-transplant lymphoproliferative disease.

Donor-derived circulating endothelial cells after kidney transplantation.

BACKGROUND: In solid-organ transplantation, the allograft vasculature, in particular the endothelium, is prone to injury inflicted by peritransplantational and posttransplantational factors. Previously, we have shown that circulating endothelial cells (cEC) can be detected in the peripheral blood of kidney allograft recipients and are often associated with acute rejection and active infections with human cytomegalovirus. In the present study we hypothesized that cEC after kidney transplantation are of donor origin, thus reflecting transplantation-related damage to the allograft endothelium. METHODS: Using hydraulic micromanipulation equipment, we isolated single cEC (n=153) from the peripheral blood of nine kidney allograft recipients at various time points after transplantation. We demonstrated the origin of these cells (donor or recipient) by typing their HLA-DRB alleles by single-cell, genomic, nested polymerase chain reaction. RESULTS: The majority (71.8%) of cEC were of donor origin and could be detected up to 141 days after onset of acute rejection episodes. Although less frequent (28.2%), recipient-type cEC were detected in the same time course as donor-type cEC. CONCLUSION: We conclude that posttransplantational injury to the allograft endothelium is reflected by the presence of donor-derived cEC in the blood.

High level expression and secretion of truncated forms of herpes simplex virus type 1 and type 2 glycoprotein D by the methylotrophic yeast Pichia pastoris.

Herpes simplex virus type 1 and 2 (HSV-1 and -2) glycoproteins D (gD-1 and gD-2) play a role in the entry of the virus into the host cell. Availability of substantial amounts of these proteins, or large fragments thereof, will be needed to allow studies at the molecular level. We studied the potency of the Pichia pastoris yeast expression system to produce soluble forms of gD. The DNA sequences encoding the extracellular domains of gD [amino acids 1-314 (gD-1(1-314)) and amino acids 1-254 (gD-1(1-254)) of gD-1 and amino acids 1-314 of gD-2 (gD-2(1-314))] were cloned into the P. pastoris yeast expression vector pPIC9. Two truncated forms of gD-1 were fitted with a His tail (designated as gD-1(1-314His) and gD-1(1-254His)) to facilitate their purification. Large amounts of gD-1(1-314) and gD-1(1-314His) (280-300mg/L induction medium) were produced. The yields of recombinant gD-1(1-254) and gD-1(1-254His) were lower: 20-36mg/L, and the yield of the gD-2(1-314) fragment was much lower: 6mg/L. SDS-PAGE analysis revealed multiple glycosylated species of the larger gD fragments, ranging in apparent molecular weight from 31 to 78kDa. The smaller gD-1(1-254) fragment appeared as two bands with molecular weights of 33 and 31kDa. All recombinant proteins produced by P. pastoris were recognized, as expected, by a panel of MAbs (A16, DL6, A18, DL11, HD1, ABDI, and AP7). In addition, we showed that gD-1(1-314), gD-2(1-314), and gD-1(1-254His) were able to interfere with binding of HSV to susceptible cells. These results indicate that the conformations of the recombinant proteins closely resemble those of native gD.

Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies.

Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1(t26-189His) and gG-2(281-594His) fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1(t26-189His) and the fragment of gG-2(281-594His) were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1(t26-189His), sera positive for HSV-2 reacted with the gG-2(281-594His) fragment, and sera positive for both types reacted with gG-1(t26-189His) and gG-2(281-594His) in Western blotting. The human sera recognized polypeptides of gG-2(281-594His) with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1(t26-189His) and the recombinant gG-2(281-594His) fragment were used as type-specific antigens for the detection of HSV-1- and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1(1-313) were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2(281-594His) fragment can be obtained in relatively large quantities at low cost.

Treatment of posttransplant lymphoproliferative disease with rituximab: the remission, the relapse, and the complication.

BACKGROUND: Rituximab, a humanized anti-CD20 monoclonal antibody, is a promising new tool for the treatment of posttransplant lymphoproliferative disease (PTLD), especially for patients transplanted with rejection prone transplants of vital organs, such as patients after lung transplantation. Thus far, no major complications have been described. We treated three lung transplant recipients with Rituximab because of PTLD. METHODS: Patients were treated with four weekly doses of 375 mg/m2 of Rituximab. Epstein-Barr virus (EBV) DNA was monitored with quantitative-competitive polymerase chain reaction and circulating B cells with flow cytometry. RESULTS: Treatment with Rituximab resulted in a complete remission in all patients without signs of or progression of bronchiolitis obliterans syndrome. Patient 1 relapsed after 2 months with a partly CD20-negative PTLD but is in stable remission after radiotherapy. Patient 2 is in complete remission 16 months after treatment, but patient 3 developed a hypogammaglobulinemia and died of invasive aspergillosis after 6 months. EBV DNA was detectable in the blood samples of patients 2 and 3 before treatment with Rituximab and became negative instantly after Rituximab. In all three patients, B cells are absent in the peripheral blood 7 months (at death), 16 months, and 16 months after treatment with Rituximab. Antiproliferating agents, such as mycophenolate mofetil (MMF), might prolong B-cell depletion. CONCLUSIONS: Rituximab was effective for the treatment of PTLD without progression of transplant dysfunction in our patients. Complications were a partly CD20-negative relapse of PTLD and a hypogammaglobulinemia. Attention should be paid to immunoglobulin G (IgG) levels, especially in patients treated with antiproliferating agents such as MMF.

Natural infection with herpes simplex virus type 1 (HSV-1) induces humoral and T cell responses to the HSV-1 glycoprotein H:L complex.

The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)H(t(His)):gL complex of HSV-1 were studied in seven HSV-1-seropositive and three HSV-1-seronegative healthy adults. In addition, responses to HSV-1 gD(t) were determined. As antigens, purified soluble recombinant forms of the gH(t(His)):gL complex produced by insect cells and of gD(t) produced by yeast cells were used. In contrast to seronegative donors, sera of all seropositive donors contained gH(t(His)): gL-specific IgG. Using peripheral blood (PB) T cells, gH(t(His)):gL-specific proliferative T cell responses were detected in all seropositive donors. Culture supernatants of PB T cells stimulated with recombinant gH(t(His)):gL contained high levels of interferon-gamma and no detectable interleukin-4, indicating their Th1 phenotype. These results show that naturally acquired HSV-1 infection induces gH:gL-specific humoral and T cell responses.