Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.     

T and B cell responses to cytomegalovirus antigens in healthy blood donors and bone marrow transplant recipients

Abstract We measured the production of interferon-gamma (IFN-γ) from single T cells and the T cell proliferative response to different cytomegalovirus (CMV) antigens in healthy blood donors and bone marrow transplant recipients. The antigens consisted of a CMV nuclear antigen (CMV na) containing the pp65-kDa matrix protein and the immediate early antigens but lacking CMV glycoproteins, and an antigen comprising native CMV glycoproteins (CMV gp). We also measured the IgG antibodies to CMV na and CMV gp. The T cells reacted to CMV na in CMV seropositive blood donors both with the production of IFN-γ and with proliferation, while bone marrow transplant recipients had a deficient T cell response. After stimulation with CMV gp, no T cell response could be observed in CMV seropositive subjects. IgG antibodies to CMV na coexisted in plasma with similar levels of antibodies to CMV gp.     

Clinical aspects of cytomegalovirus infection after allogenic bone marrow grafts

CMV infection is one of the major infection after bone marrow transplantation. CMV viremia was systematically studied in 66 patients with aplastic anemia or leukemia undergoing BMT. 57% patients had CMV viremia with a frequency peak between 7 and 9 weeks after transplant. Clinical symptoms found during viremia were pancytopenia, fever, cytolytic hepatitis. Interstitial pneumonitis was found only in 4 cases. In 3 cases, viremia was not associated with clinical symptoms. Survival was identical to the group of patients without viremia. Viremia was positively associated with the presence of high anti-CMV antibody titer in donor or recipient before transplant, or to a lymphocyte proliferative response against CMV antigens in donor or recipient before BMT. Granulocyte transfusions increased the frequency of CMV viremia. CMV infection was significantly associated with acute and chronic graft versus host disease. The relation showed between these parameters and viremia provides a basis for an accurate diagnosis of CMV infection and a better background for the study of prophylactic or curative treatment of CMV infection.     

CMV infections in bone marrow transplanted patients--evaluation of prophylaxis with Cytotect (CMV hyperimmuneglobulin)

Cytomegalovirus (CMV) infection is a frequent and clinically important infection following bone marrow transplantation. Candidates for this study were patients admitted for transplantation: 22 patients received bone marrow from a HLA-identical, MCR-nonreactive sibling, in 9 patients an autologous BMT was performed. The anti-CMV IgG (Cytotect) was administered at a dosage of 1 ml/kg on days -7, 13, 33, 53, 73 and 93 after BMT. 5 patients in the very beginning of our BMT program did not receive Cytotect. Patients were given random blood products from the bloodbank not tested for CMV positivity. Active CMV infection or seroconversion in our patients was defined as a rise in IgG titer against the late antigen of fourfold or more or an IgM increase. In the allogeneic BMT group the pretransplant serological status was in 6 cases negative in recipients and donor, in 7 patients positive in recipients and negative in donors, and in 4 patients positive in recipients and donors. Of the 6 patients seronegative in recipients and donors, 3 developed active infection and of the 7 patients pretransplant positive with seronegative donors 3 developed active infection and 4 latent infections during the period from 2 to 100 days following grafting. 1 patient out of the group transplanted in third partial remission of AML developed interstitial pneumonia and died on day +30.4 of the 4 cases with seropositivity of recipients and donors developed active CMV infection. Of 9 patients with autologous transplantation 6 patients were pretransplant seropositive. 3 of these 6 developed active infection and 2 latent infection 30 to 180 days after grafting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of cytomegalovirus in pig-to-primate organ xenotransplantation

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.     

Replication of herpes simplex virus and cytomegalovirus in human leukocytes.

Human peripheral blood leukocytes, lymphocyte subpopulations, and hemic cell lines were examined for their ability to supprot HSV and CMV replication. Mitogen-stimulated mononuclear leukocytes, B lymphocytes, and T lymphcytes supported the replication of HSV to high titers over 3 to 5 days of infection. HSV replicated in unstimulated mononuclear leukocyte cultures of one of five donors, and to a limited degree in untreated B lymphocytes of three of five donors; HSV replication was not detected in unstimulated T lymphocytes (five donors). There was no evidence of enhanced uptake of 3H-thymidine in the untreated donor cells that replicated HSV. CMV replication was not detected during 9 to 10 days of infection in untreated or mitogen-treated mononuclear leukocytes and lymphocyte subpopulations from the same adult donors or in neonatal cord blood leukocytes. The ability of the cells to support HSV or CMV replication did not correlate with the presence of specific antiviral antibodies in the donor serum. HSV replication in B, T, and myeloid cell lines to high titers over 5 days of infection, whereas CMV failed to replicate in any of the hemic cell lines. A persistent HSV infection has been established in a T cell line (CEM) with high titers of infectious virus being produced concurrently with growth of the cells over the first 11 weeks of infection.     

Cytomegalovirus-mediated upregulation of chemokine expression correlates with the acceleration of chronic rejection in rat heart transplants

Cytomegalovirus (CMV) infections have been shown to dramatically affect solid organ transplant graft survival in both human and animal models. Recently, it was demonstrated that rat CMV (RCMV) infection accelerates the development of transplant vascular sclerosis (TVS) in both rat heart and small bowel graft transplants. However, the mechanisms involved in this process are still unclear. In the present study, we determined the kinetics of RCMV-accelerated TVS in a rat heart transplant model. Acute RCMV infection enhances the development of TVS in rat heart allografts, and this process is initiated between 21 and 24 days posttransplantation. The virus is consistently detected in the heart grafts from day 7 until day 35 posttransplantation but is rarely found at the time of graft rejection (day 45 posttransplantation). Grafts from RCMV-infected recipients had upregulation of chemokine expression compared to uninfected controls, and the timing of this increased expression paralleled that of RCMV-accelerated neointimal formation. In addition, graft vessels from RCMV-infected grafts demonstrate the increased infiltration of T cells and macrophages during periods of highest chemokine expression. These results suggest that CMV-induced acceleration of TVS involves the increased graft vascular infiltration of inflammatory cells through enhanced chemokine expression.     

Shedding light on the elusive role of endothelial cells in cytomegalovirus dissemination

Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2-cre ⁺ heart and kidney transplants were readily transmitted to MCMV-naïve recipients by primary viremia. In contrast, when a Tie2-cre ⁺ transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre ⁺ recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.     

Immunostimulation by cytomegalovirus (CMV): helper T cell-dependent activation of immunoglobulin production in vitro by lymphocytes from CMV-immune donors

Cytomegalovirus (CMV) is the cause of a number of different diseases ranging from self-limited benign infections in healthy adults to life threatening illnesses among immunocompromised hosts and newborns. Suppression of cell-mediated immunity is often found in cases of acute CMV infection, and in addition, the virus may also be a potent stimulant of lymphoid cells in vivo. We studied cellular proliferation and immunoglobulin (Ig) production induced by CMV to determine its effect on human lymphocytes in vitro. The CMV that was added to cultures of lymphocytes from CMV-seronegative donors failed to induce either significant cellular proliferation or Ig production. By contrast, CMV-stimulated cultures from CMV-seropositive donors induced both prominent cellular proliferation and Ig production. B cell differentiation into Ig-secreting cells required the presence of T cells, and this T cell help was sensitive to irradiation with 2000 rad and to treatment with cyclosporin A. When T cells were depleted of OKT4+ cells with monoclonal antibody and complement, the co-cultured B cells failed to produce Ig, whereas the depletion of OKT8+ cells had no effect on the Ig-secreting cell response. Inactivation of CMV before culture did not result in a reduction of either cellular proliferation or Ig production. Thus, infection of target cells is not required for in vitro lymphocyte activation by CMV. These results demonstrate that CMV is a potent activator of B cells inducing Ig production in vitro, and that this process requires the presence of virus-specific memory T cells.     

Lungs are a major organ site of cytomegalovirus latency and recurrence.

Recurrence of infectious virus from the latent viral genomes is the initiating event in the pathogenesis of cytomegalovirus (CMV) disease during states of immunodeficiency. Interstitial pneumonia is a frequent manifestation of posttransplantation CMV disease, in particular after bone marrow transplantation and heart and lung transplantations. Recurrence can occur within the transplant derived from a latent infected donor as well as within latently infected organs of the transplant recipient. The reason for a predilection of the lungs as a site of CMV pathology is so far unknown. In a murine model of CMV latency, the lungs were identified as an authentic site of latent infection, since the viral genome remained detectable in lung tissue even after it was cleared to an undetectable level in blood and bone marrow. A comparison between the lungs and the spleen, the previously most thoroughly investigated site of murine CMV latency, revealed a 10-fold-higher burden of latent viral genome for the lungs. Most important, the organ-specific risk of in vivo recurrence was found to correlate with the organ-specific viral genomic load. This new finding thus characterizes the lungs as a high-risk organ for CMV recurrence, and this fact may explain in part why interstitial pneumonia is a frequent manifestation of recurrent CMV infection.     

Host and donor immune responses contribute to antiviral effects of amotosalen-treated donor lymphocytes following early posttransplant cytomegalovirus infection

We have previously shown that amotosalen-treated splenocytes rescued allorecipients from a lethal dose of mouse CMV (MCMV) administered on day 0 in experimental parent C57BL/6-->CB6F1 allogeneic bone marrow transplant. In this study, we investigated the mechanism of antiviral activity of amotosalen-treated donor splenocytes when sublethal MCMV infections were administered 7 days posttransplant. Recipients of 3 x 10(6) untreated splenocytes were used as control. Following MCMV infection, recipients of untreated splenocytes had 40% early mortality due to acute graft-vs-host disease compared with no deaths among recipients of 10 x 10(6) treated splenocytes. However, recipients of both types of donor splenocytes effectively cleared MCMV from their liver. Like the untreated CD8(+) T cells, amotosalen-treated CD8(+) T cells equally retained their in vivo CTL activity against MCMV early peptide-pulsed targets and expressed similar levels of granzyme B within 11 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with both donor spleen- and host-derived anti-MCMV CD8(+) T cells in their blood and lymphoid organs, with significantly higher numbers of host-derived CD4(-)CD8(-) (double negative) T cells in the spleens of recipients of treated splenocytes compared with the recipients of untreated splenocytes. Additionally, recipients of amotosalen-treated splenocytes had lower levels of serum IFN-gamma and TNF-alpha in response to MCMV infection compared with untreated recipients. Thus, adoptive immunotherapy with treated T cells is a novel therapeutic approach that facilitates hematopoietic engraftment and permits antiviral immunity of both donor and host T cells without graft-vs-host disease.     

Identification of cytomegalovirus (CMV) infection by different laboratory methods in renal transplant recipients undergoing triple-drug immunosupressive treatment.

Early diagnosis of CMV infection is very important mainly in transplant recipients because CMV infection is a frequent complication after transplantation. In this work we compared different laboratory methods: ELISA (IgG, IgM), Western blot,shell vial, antigenemia assay (pp65), the immunofluorescent method with epithelial cells from urine (IF), DNA in leukocytes by PCR and DNA in leukocytes by hybridization (HCS) to estimate the most proper method for diagnosis of CMV in renal transplant recipients. This preliminary study showed that HCS, PCR and Western blot are sensitive methods for detecting CMV infection. Using HCS in quantitative variant we obtained a very good correlation between DNA load and clinical symptoms.     

Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus,Epstein-Barr virus,and polyomavirus BK in whole blood from transplant candidates

Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR) method to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R² = 0.997) and reproducibility (CV range, 0.95–2.38%, 0.52–3.32%, and 0.31–2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.     

Changes in cell-mediated immunity in kidney transplant recipients with active CMV infection

This study was aimed at determining (a) the extent of proliferation of peripheral blood mononuclear cells (PBMC) in response to stimulation by cytomegalovirus (CMV)-infected fibroblasts and (b) the levels of Th1 and Th2 cytokine production in kidney transplant recipients with and without active CMV infection. Thirty patients with, and 39 without active CMV infection, diagnosed by a CMV antigenemia assay (AA), were studied. PBMC of patients with active CMV infection showed significantly lower proliferation than those without ongoing CMV infection (P<0.0001). The levels of Th2-type cytokines (interleukin (IL-) 4 and IL-10) in AA-negative and AA-positive kidney transplant recipients were similar but the levels of the Th1-type cytokines interferon-gamma, tumor necrosis factor-alpha (P<0.05) and IL-2 were significantly lower in AA-positive kidney transplant recipients (P<0.0005).     

Bone marrow transplantation in a patient with drug-induced aplastic anemia

A 23-year-old woman gravely ill with Pseudomonas septicemia secondary to presumed drug-induced bone marrow aplasia received marrow transplantation from two male HL-A identical sibling donors. She had a successful engraftment with excellent but temporary clinical improvement. Subsequently she succumbed to graft-versus-host disease manifested by Pseudomonas and Candida albicans septicemia, cytomegalovirus pneumonitis, three phases of dermatitis, nausea, vomiting, dysphagia, diarrhea, fever, edema and bone pain, with gradual but complete graft suppression by the 74th day after the transplantation. A second marrow transplant on the 70th day was unsuccessful.     

Requirements for the adoptive transfer of antibody responses to a priming antigen in man

Antibody responses to recall vaccines can be adoptively transferred after marrow transplantation in man. Transfer of responses to priming Ag has not been successful, although this would broaden the range of organisms to which recipients could be protected. To investigate the importance of T cells and Ag in such transfer we primed marrow donors with keyhole limpet hemocyanin (KLH) 1 or 3 wk before marrow harvesting. B cells secreting IgM and IgG anti-KLH antibody were present in donor marrow at both 1 and 3 wk after immunization. After T cell depletion, donor marrow was infused into chemo-irradiated recipients, half of whom were immunized pretransplant with KLH. We found no evidence for the transfer of the IgM component of the response. Clonal expansion of the transferred IgG antibody-secreting cells with a corresponding rise in recipient serum IgG antibody levels was seen only when donors were primed 3 wk before marrow harvest and when the recipients were also immunized. IEF and immunoblotting demonstrated that successful transfer coincided with maturation of the IgG primary response from a polyclonal to an oligoclonal pattern and confirmed that donor oligoclonal bands appeared in the recipient serum. We conclude that the immunization protocols required for the transfer of antibody responses to priming Ag reflect the initial dependence of unprimed B cells on T cell help and on prolonged Ag stimulation. Ag-stimulated primary B cells in T cell-depleted marrow respond only to the noncognate growth and differentiation signals available in the chemo-irradiated recipient after an initial period of clonal selection and expansion in the donor which is both T cell and Ag dependent. Even after this initial selection, continued expansion of antibody-secreting clones in recipients retains an absolute dependence on Ag stimulation. Immunization techniques to protect transplant recipients against organisms such as Pseudomonas and CMV may need to be modified accordingly.     

Use of acyclovir in the prevention of herpes infections after allogenic bone marrow grafts

In a double-bind controlled study, oral Acyclovir has been compared to a placebo in a series of 39 consecutive patients undergoing bone marrow transplantation. A dose of 200 mg was given every 6 h from day 8 to day 35 after transplantation. Pharmacokinetic studies have shown the good absorption of the drug despite intestinal damage related to chemoradiotherapy or gut graft-versus-host disease (GVHD), there was no sign of toxicity. The protection against herpes simplex virus (HSV) infection was complete in the treated group when compared to the control group even in patients with high anti-HSV antibody titres. The same protection was observed against cytomegalovirus (CMV) infection. The incidence of HSV and CMV was the same in both groups after treatment ended. This study confirms the efficacy of Acyclovir against HSV infection and possibly against CMV infection when it is given prophylactically after bone marrow transplantation.     

Serum antibodies to individual cytomegalovirus structural polypeptides in renal transplant recipients during viral infection

In a longitudinal study we examined by immunoblotting (IB) the development and the evolution of the humoral immune response against individual cytomegalovirus (CMV) structural polypeptides in a total of 80 serum samples from 13 renal transplant recipients showing serological evidence of CMV infection and five renal transplant recipients with an anti-CMV antibody level unchanged over the observation period. The results showed that the IB reactivity at the time of transplantation may be a good index of the host's humoral immune status against CMV; by using this procedure it is possible to identify a seroconversion by the detection of antibodies reacting with some intermediate molecular weight proteins in sera examined at high dilution. Furthermore, IB is a very sensitive procedure also for IgM detection as it anticipates the positivity of the enzyme immune assay for IgM.     

Oral Valganciclovir as a Preemptive Treatment for Cytomegalovirus (CMV) Infection in CMV-Seropositive Liver Transplant Recipients

Objectives

Cytomegalovirus (CMV) infections in liver transplant recipients are common and result in significant morbidity and mortality. Intravenous ganciclovir or oral valganciclovir are the standard treatment for CMV infection. The present study investigates the efficacy of oral valganciclovir in CMV infection as a preemptive treatment after liver transplantation.

Methods

Between 2012 and 2013, 161 patients underwent liver transplantation at Samsung Medical Center. All patients received tacrolimus, steroids, and mycophenolate mofetil. Patients with CMV infection were administered oral valganciclovir (VGCV) 900mg/day daily or intravenous ganciclovir (GCV) 5mg/kg twice daily as preemptive treatment. Stable liver transplant recipients received VGCV.

Results

Eighty-three patients (51.6%) received antiviral therapy as a preemptive treatment because of CMV infection. The model for end-stage liver disease (MELD) score and the proportions of Child-Pugh class C, hepatorenal syndrome, and deceased donor liver transplantation in the CMV infection group were higher than in the no CMV infection group. Sixty-one patients received GCV and 22 patients received VGCV. The MELD scores in the GCV group were higher than in the VGCV group, but there were no statistical differences in the pretransplant variables between the two groups. AST, ALT, and total bilirubin levels in the GCV group were higher than in the VGCV group when CMV infection occurred. The incidences of recurrent CMV infection in the GCV and VGCV groups were 14.8% and 4.5%, respectively (P=0.277).

Conclusion

Oral valganciclovir is feasible as a preemptive treatment for CMV infection in liver transplant recipients with stable graft function.     

Cytomegalovirus Inhibits the Engraftment of Donor Bone Marrow Cells by Downregulation of Hemopoietin Gene Expression in Recipient Stroma

Cytomegalovirus (CMV) disease after bone marrow (BM) transplantation is often associated with BM graft failure. There are two possible reasons for such a correlation. First, a poor hematopoietic reconstitution of unrelated etiology could promote the progression of CMV infection by the lack of immune control. Alternatively, CMV infection could interfere with the engraftment of donor BM cells in recipient BM stroma. Evidence for a causative role of CMV in BM aplasia came from studies in long-term BM cultures and from the murine in vivo model of CMV-induced aplastic anemia. A deficiency in the expression of essential stromal hemopoietins, such as stem cell factor (SCF), has indicated a functional insufficiency of the stromal microenvironment. It remained open to question whether CMV mediates a negative regulation of hemopoietin gene expression (the downregulation model) or whether it causes the default of a positive regulator (the lack-of-induction model). Further, even though implicitly assumed, it has never been formally documented that CMV directly interferes with the engraftment of a BM cell transplant. We addressed these problems in a murine model of CMV infection after experimental male-into-female BM transplantation. The data indicate that the downregulation model applies. Quantitation of the male-sex-determining gene tdy demonstrated an impaired engraftment of donor BM cells in the BM stroma of the female recipients. This graft failure was reflected by a diminished population of SCF-receptor-expressing hematopoietic progenitor cells and correlated with a reduced level of stromal SCF gene expression. Interestingly, high doses of BM cells protected against stromal insufficiency by a mechanism unrelated to control of infection.