PDGFR-Mediated Cytomegalovirus Infection of Megakaryocytes Induces Thrombocytopenia Via Demethylation of AML1 and IEX-1 in the TPO/c-Mpl Signaling Pathway Following Allo-HSCT

Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Cytomegalovirus Infection in Mesenchymal Stem Cells and Their Activation Could Be Enhanced by Nuclear Factor-κB Inhibitor Pyrrolidinedithiocarbamate In Vitro

Cytomegalovirus Infection in Bone Marrow-Derived Mesenchymal Stem Cells: Implications for Functions and Exploration of Relevant Mechanisms

Comparing Costs Associated to Letermovir Prophylaxis Vs Ganciclovir Pre-Emptive Therapy in HCMV-Seropositive Patients Who Undergone to Hemopoietic Stem Cells Transplantation

Human cytomegalovirus (HCMV) infections are common and lead to lifelong infections. In immunocompetent individuals, primary infections are mostly subclinical or they may be associated with a self-limited mononucleosis-like syndrome. In contrast, infections in immunocompromised hosts (either primary infections, reactivations from latency, or reinfections) are associated with important morbidity and mortality. In patients with AIDS, the introduction of highly active antiretroviral therapy (HAART) has decreased the overall incidence of HCMV disease, mainly retinitis and gastrointestinal infections, by about 80% (50, 105). However, the functional benefit of HAART (i.e., restoration of specific HCMV-specific immune responses) may take up to 3 to 6 months to occur, and some patients do not have access to or do not respond to HAART (3, 40, 67, 104). Thus, HCMV still remains a concern in AIDS patients with CD4 counts <50 to 100 cells/μl. A specific HCMV syndrome consisting of fever, malaise, arthralgia, and neutropenia may occur in solid-organ transplant (SOT) patients, in particular, those developing a primary HCMV infection (i.e., HCMV-seronegative recipient from a HCMV-seropositive donor [D+/R−]) during the first 3 months posttransplantation. In addition, invasive HCMV disease may involve different organs, such as the lungs, liver, and gastrointestinal tract. In the absence of antiviral intervention, symptomatic HCMV infections occur in approximately 39 to 41% of heart-lung transplant recipients, 9 to 35% of heart transplant recipients, 22 to 29% of liver and pancreas transplant recipients, 8 to 32% of kidney transplant recipients, 50% of kidney-pancreas transplant recipients, and 22% of small-bowel transplant recipients (114), with the highest incidence seen in D+/R− patients. Finally, active HCMV infections have been associated with indirect effects, such as dysfunction or rejection of the transplanted organ, an increased risk for bacterial or fungal opportunistic infections, and accelerated atherosclerosis in heart transplant recipients (109). Among allogeneic bone marrow transplant (BMT) or hematopoietic stem cell transplant (HSCT) recipients, pneumonia and enteritis are the most common clinical manifestations of HCMV disease. In HCMV-seropositive recipients, active HCMV infections occur in 70 to 80% of patients; and in the absence of antiviral intervention, disease develops in 20 to 35% of those individuals, whereas active infections occur in only 15% of seronegative recipients of marrow from a seropositive donor (102). HCMV pneumonia remains associated with a significant risk of mortality, even when specific antiviral treatment is administered (18, 101). Gastrointestinal disease, alone or in association with pulmonary disease, is the second most common clinical manifestation of HCMV infections in that setting (85). In addition to AIDS patients and transplant recipients, HCMV infections have been associated with serious complications in other immunocompromised hosts, such as cancer patients, mostly those suffering from hematologic malignancies (48), and children with congenital primary immunodeficiencies (135). Finally, congenital HCMV infections can result in severe sequelae in newborns or during the first years of life (22).

Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells.&#13;\nBased on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development.&#13;\nFunctional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner. &#13;\nIn summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms.

First-Line Therapy with Donor Derived HCMV-Specific T Cells Reduce Persistent HCMV Infection after Allogenic Stem Cell Transplantation

Human cytomegalovirus infection of cells of hematopoietic origin: HCMV-induced immunosuppression, immune evasion, and latency

Expanded clinical-grade NK cells exhibit stronger effects than primary NK cells against HCMV infection.

Evidence suggests that human cytomegalovirus (HCMV) infection may be implicated in the progression of colorectal cancer (CRC). However, the correlation between HCMV infection and survival outcomes in patients with CRC remains unclear. Here, we constructed a flow algorithm to identify HCMV sequences based on the RNA-seq data of patients with CRC derived from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The patients' clinical information matrix was used to calculate the Euclidean distance to filter out suitable patients not infected with HCMV, combined with patients' survival outcomes, to reveal how HCMV infection is involved in CRC progression. HCMV infection is widespread in patients with CRC, and the prevalence of HCMV infection ranges from 10 to 36% in four independent CRC datasets, with infection being concentrated in carcinoma tissue rather than in normal tissue. In addition, HCMV-positive patients had a poor survival prognosis, with three HCMV genes, UL82, UL42, and UL117, associated with poor patient survival outcomes. Most importantly, we suppose that the regulation of immune function by HCMV may be key to the poor prognosis of patients with CRC. We found that HCMV infection was associated with poor prognosis in CRC patients and identified three prognosis-associated HCMV genes. The regulation of immune function caused by HCMV infection was the key factor, while HCMV-positive patients with CRC mostly presented with a state of immunosuppression. This may provide new ideas for the personalized treatment of patients with CRC, especially with respect to immunotherapy.

In immune-competent individuals, human cytomegalovirus (HCMV) infection is associated with impairment of T-cell function. Our goal was to evaluate prospectively whether clinically asymptomatic HCMV infection in allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients, treated pre emptively with ganciclovir, influences T-cell function as well. Mitogen-stimulated T-cell proliferative activity, together with cell surface markers, was tested in 49 patients on days + 30, + 45, + 60, and + 90 after alloHSCT and, additionally, in cases of positive HCMV pp65-antigenemia. HCMV infection was diagnosed in 19 patients. None of them developed HCMV disease. T-cell proliferative activity was significantly decreased on days when HCMV antigenemia was positive as compared to days without antigenemia. The number of pp65-positive cells negatively correlated with proliferative response. Comparison of patients who did experience HCMV infection with those who did not reveals significant decrease of T-cell proliferative activity observed on days + 30 and + 45, a time period when antigenemia was most frequently found to be positive, whereas no difference was detected on days + 60 and + 90. We conclude that, even clinically asymptomatic, HCMV infection has negative impact on T-cell proliferation capacity in alloHSCT recipients. However, pre emptive therapy with ganciclovir makes this immunosuppressive effect transient and restricted to the time of infection duration.

Objective To observe the changes in the expression of pro-inflammatory cytokines IL-1β over time in THP-1 derived macrophages after human cytomegalovirus (HCMV) infection. Methods The model of HCMV infection in THP-1 derived macrophages was constructed, then HCMV infected group, the mock-infected control group, LPS + ATP group and poly (dA : dT) control group were established. ELISA analysis was performed to measure the levels of IL-1β in culture supernatant of THP-1 derived macrophages at 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours after infected with HCMV. Real-time PCR and western blot were performed to measure the expression levels of IL-1β gene and protein, respectively. Results The gene expression of pro-inflammatory cytokines IL-1β in HCMV infected group is 7.77 times to the mock-infected control group at 6 hours after HCMV infected THP-1 derived macrophages. IL-1β in supernatant of THP-1-derived macrophages increased significantly at 1 hour after HCMV infection, was continuously rising at 3 hours and 6 hours subsequently, and reached at peak at 12 hours and maintained till 48 hours. The protein expression of IL-1β in HCMV infected group was significantly higher than the mock-infected control group and poly (dA : dT) control group at 6 hours after HCMV infected THP-1 derived macrophages, while no significant difference between the LPS + ATP control group. Conclusions The expression of IL-1β is induced by HCMV infected THP-1 derived macrophages, and continue to increase over time. Key words: Human cytomegalovirus; THP-1 derived macrophages; Interleukine-1 beta

Human cytomegalovirus (HCMV) infection is usually implicated in thrombocytopenia occurring in newborns and immunocompromised patients. However, the underlying mechanisms remain elusive. This study was conducted to investigate the effects of HCMV infection on the viability of megakaryocytic CHRF-288-11 cells and the underlying mechanisms involved. RT-PCR for determining mRNA expression of HCMV immediate early gene 1 and Western blot for measuring protein expression of late HCMV gene pp65 showed that CHRF-288-11 cells were susceptible to HCMV infection. HCMV infection reduced the viability of CHRF-288-11 cells via apoptosis in a dose- and time-dependent manner. Both caspase 3 and c-Jun terminal kinase (JNK) signaling pathway were activated in the HCMV-treated CHRF-288-11 cells. z-DEVD-fmk (a caspase inhibitor) and SP600125 (a JNK inhibitor) significantly prevented the death of CHRF-288-11 cells induced by HCMV, respectively. Furthermore, inhibition of JNK activity could reduce the formation of active caspase 3 induced by HCMV. Interestingly, the co-application of antivirus drug ganciclovir and SP600125 synergistically prevented the death of CHRF-288-11 cells induced by HCMV. Collectively, these findings suggest that HCMV infection may induce the caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by the activation of JNK signaling pathway.

Thrombocytopenia is a common complication of human cytomegalovirus (HCMV) infection in immunocompromised hosts, which contributes to poor prognosis even in patients receiving antiviral treatment. Here, we investigated the megakaryo/thrombopoiesis process, including the involvement of the c-Mpl/IEX-1 pathway, after HCMV infection, identified receptors mediating the interaction between megakaryocytes (MKs) and HCMV, and explored novel therapeutic targets. Our data shows that HCMV directly infects megakaryocytes in patients with HCMV DNAemia and influences megakaryopoiesis via the c-Mpl/IEX-1 pathway throughout megakaryocyte maturation, apoptosis, and platelet generation in vivo and in vitro. After treatment with inhibitors of PDGFRα and αvβ3, the HCMV infection rate in MKs was significantly reduced, suggesting that IMC-3G3 and anti-αvβ3 are potential therapeutic alternatives for viral infection. In summary, our study proposes a possible mechanism and potential treatments for thrombocytopenia caused by HCMV infection and other viral diseases associated with abnormal hemostasis.

Human cytomegalovirus (HCMV) infection may cause substantial morbidity and mortality after renal and bone marrow transplantation [1]. There are 3 major consequences of HCMV infection: HCMV disease with a wide range of clinical illnesses; superinfection with opportunistic pathogens; and injury to the transplanted organ [2]. Other than serological method to diagnose HCMV infection, viral load quantitation by real time polymerase chain reaction has been widely appreciated to diagnose and monitor the progress of viral infections. The aims of this study were to determine the incidence of HCMV infection in renal and bone marrow transplant recipients and to investigate its associations with HCMV disease, gender, and races.&#x0D; This retrospective cohort analysis involved 1520 blood samples from transplant recipients (renal, n = 164 and bone marrow, n = 182) from January 2020 to December 2021 collected from Virology Unit, Hospital Kuala Lumpur (NMRR ethical approval: NMRR-20-993-53201(IIR). The samples were analysed with quantitative polymerase chain reaction for HCMV DNA and the demographic, clinical and paraclinical aspects were evaluated. HCMV infection was present if the patient had positive HCMV viraemia and HCMV disease was diagnosed if HCMV infection was followed by clinical signs and symptoms. Statistical comparisons of patient demographics were performed with Chi-square tests for the categorical variables.&#x0D; The overall incidence of HCMV infection in the study group was 65% (225/346) where renal and bone marrow transplants account for 78.2% (176/225) and 21.7% (49/225) respectively. The incidence of HCMV infection in renal transplantation differed significantly by sex (p&lt;0.05) where it was higher in males (71.8%) than in females (28.2%) but there was not statistically significant by sex in bone marrow transplantation in which males and females account for 61.2% and 38.7% respectively. The incidence of HCMV differed significantly (p&lt;0.05) by races in both transplantation types as follows: 58% in Malay, 36% in Chinese, 5% in Indian and 1% in other indigenous races in renal transplantation while 59% in Malay, 29% in Chinese, 10% in Indian and 2% in other indigenous races in bone marrow transplantation. The incidence of HCMV disease differed significantly (p&lt;0.05) by type of transplantation where it is higher in renal transplantation (30.9%) than in bone marrow transplantation (20.2%). The most seen symptoms were fever, generalised lethargy, and headache. Viral load of HCMV has been shown to be a major determinant factor in the severity and the manifestation of the HCMV infection[3].&#x0D; It is significantly higher in patients who develop HCMV disease[3]. Various risk factors have been described for the progress of symptomatic HCMV infection in organ transplant recipient[3]. The incidence of HCMV infection was higher in renal transplant as compared to bone marrow transplant among Malaysian. This study has shown that HCMV viral load has a significant association with age, gender and HCMV disease. Various syndromes can be caused by HCMV ranging from a mild fever to severe end-organ diseases. Treatment with anti-HCMV therapy results in decline in HCMV load, usually to undetectable.