Pp65 Antigen Research Articles

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.

BackgroundReactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCpp65”) that accelerated antigen-specific T cell development.MethodsHere we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed.ResultsUpscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated.ConclusionThese results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0599-5) contains supplementary material, which is available to authorized users.

Study on the correlation between CMV reactivation and bronchiolitis obliteans after allogeneic hematopoietic stem cell transplantation

目的探讨异基因造血干细胞移植（allo-HSCT）术后CMV重激活与闭塞性细支气管炎（BO）的相互关系。方法2011年1月至2013年12月行allo-HSCT的769例患者，通过荧光定量PCR方法检测CMV-DNA水平，免疫荧光染色白细胞PP65抗原血症两种检测方法诊断CMV感染，将CMV感染患者出现BO与未发生CMV感染患者出现BO进行比较，分析CMV感染与BO相关性，并将符合CMV感染患者中出现BO与未出现BO者进行对照分析。结果诊断CMV感染259例患者中出现BO 32例（12.35%），而未感染CMV的510例患者发生BO 8例（1.56%），二者差异有统计学意义（P< 0.001）。将诊断CMV感染259例患者，发生BO 32例与未发生BO 227例的二组临床资料进行CMV相关临床分析比较。CMV病毒载量在105拷贝数／ml较低病毒载量102拷贝数／ml出现BO差异有统计学意义。结论在allo-HSCT后导致BO的风险因素中，CMV感染是值得注意的相关因素之一，其中高病毒载量和CMV重激活和CMV肺炎是可能危险因素。

456. Pharmacodynamics and GMP Development of a Lentivirus-Induced DC Vaccine for Accelerated Adaptive Immune Reconstitution Against Cytomegalovirus after Stem Cell Transplantation

Effective immunization of patients shortly after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an unmet clinical need due to delayed de novo development of T and B cells. We demonstrated a recombinant, donor-derived cell vaccine against cytomegalovirus that can be easily manufactured, cryopreserved and, upon thaw and administration, turn themselves into highly-viable dendritic cells (DCs). The cell source consisted of monocytes from peripheral blood or cord blood (CB), transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65). The pharmacodynamics of the frozen/thawed vaccine (“SmyleDCpp65”) was evaluated by immunization of NOD.Rag1–/–.IL2rγ–/– mice transplanted with human CD34+ CB stem cells. SmyleDCpp65 stimulated in vivo thymic and extrathymic expansion of human cytotoxic T cells (CTL) reactive against pp65, and high levels of human immunoglobulins and immune-stimulatory cytokines in plasma. Standardized production of the IDLV and SmyleDCpp65 was established using Good Manufacturing Practices (GMP). Analytical parameters for quality control demonstrated high recovery, uniformity, purity and viability of SmyleDCpp65 after thawing. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to protooncogenic hot-spots. These results underscore further developments of this individualized cell vaccine to accelerate immune reconstitution against cytomegalovirus after allo-HSCT.

524. Immune-Monitoring of Mature Human T Cell Reconstitution in Humanized NRG Mice After Cord Blood Stem Cell Transplantation and Engineered Dendritic Cell Prime/Boost: TCR Polyclonality and No GvHD

Cord blood (CB) is a major and convenient “off the shelf” source of stem cells for hematopoietic stem cell transplantation (HSCT) when a matched donor is not available. Although relatively lower incidence of graft versus host disease is observed after CB-HSCT than PB-HSCT, delayed T cell immune reconstitution, relapse and viral infections harmfully impact survival after CB-HSCT. We validated genetically reprogrammed CB-derived monocytes capable to differentiate into dendritic cells in vivo as means to improve human immune reconstitution in lymphopenic hosts and protect them against cytomegalovirus (CMV). Monocytes were isolated from the CB CD34− fraction, transduced with an integrase-defective lentiviral vector (co-expressing GM-CSF, IFN-α and the CMV antigen pp65) and administered after CB-HSCT or cryopreserved. Prime/boost administrations of the engineered “SmyleDCpp65” cell vaccine into NOD.Rag1–/–.IL2rγ–/– mice 6-11 weeks after CB-HSCT resulted into higher levels of human T cell development in the thymus and evidence of maturation of memory and effector T cells in peripheral blood and secondary lymphatic tissues. This model allows mechanistic characterization of human CTL, Th and Treg cell reconstitution (by multicolor flow cytometry), homing (bone marrow, thymus, spleen, lymph nodes, liver; by flow cytometry), immune functions (pp65-specific responses by ELISPOT and cytotoxic assays), T cell receptor polyclonality analyses (by deep sequencing), inflammatory responses (array analyses of plasma cytokines and immune globulins). Despite the de novo activation of human T cells, GVHD was not observed. This model demonstrates the prof-of-concept of an adjuvant cell vaccine to be used in conjunction with CB-HSCT in the clinics and offers a practical experimental system to study adaptive human T cell immune reconstitution and antigen-specific responses in vivo. We are currently evaluating further approaches to maximize lentiviral transduction of CB monocytes and co-stimulation of human antibody responses against the cytomegalovirus gB antigen in the CB-HSCT model.

Diversified phenotype of antigen specific CD8+ T cells responding to the immunodominant epitopes of IE and pp65 antigens of human cytomegalovirus

To study the cytomegalovirus (CMV)-specific CD8+ T cells in individuals with HLA A∗1101, A∗0201 and A∗2402, our findings showed that peptide SK-10-2, KI-10 and KV-10 of CMV IE and pp65 antigens were immunodominant in 198 individuals with HLA A∗1101, A∗0201 and A∗2402, the most frequent genotypes in Chinese. Interestingly, SK-10-2 induced the strongest T cell response to produce IFN-γ whereas the others did not induce prominent IFN-γ production despite they all induced remarkable T cell proliferation. The peptides induced different phenotypes including IFN-γhighTNF-αlow and TNF-αlowFoxp3low. It suggests that only some of CMV-reactive CD8+ T cells are real protective IFN-γhigh cytotoxic T cells.

High Reproducibility of ELISPOT Counts from Nine Different Laboratories.

The primary goal of immune monitoring with ELISPOT is to measure the number of T cells, specific for any antigen, accurately and reproducibly between different laboratories. In ELISPOT assays, antigen-specific T cells secrete cytokines, forming spots of different sizes on a membrane with variable background intensities. Due to the subjective nature of judging maximal and minimal spot sizes, different investigators come up with different numbers. This study aims to determine whether statistics-based, automated size-gating can harmonize the number of spot counts calculated between different laboratories. We plated PBMC at four different concentrations, 24 replicates each, in an IFN-γ ELISPOT assay with HCMV pp65 antigen. The ELISPOT plate, and an image file of the plate was counted in nine different laboratories using ImmunoSpot® Analyzers by (A) Basic Count™ relying on subjective counting parameters set by the respective investigators and (B) SmartCount™, an automated counting protocol by the ImmunoSpot® Software that uses statistics-based spot size auto-gating with spot intensity auto-thresholding. The average coefficient of variation (CV) for the mean values between independent laboratories was 26.7% when counting with Basic Count™, and 6.7% when counting with SmartCount™. Our data indicates that SmartCount™ allows harmonization of counting ELISPOT results between different laboratories and investigators.

Detection of human cytomegalovirus in different histopathological types of glioma in Iraqi patients.

Human Cytomegalovirus (HCMV) is an endemic herpes virus that reemerges in cancer patients enhancing oncogenic potential. HCMV infection is associated with certain types of cancer morbidity such as glioblastomas. HCMV, like all other herpes viruses, has the ability to remain latent within the body of the host and can contribute in chronic inflammation. To determine the role of HCMV in glioma pathogenesis, paraffin-embedded blocks from glioma patients (n = 50) and from benign meningioma patients (n = 30) were obtained and evaluated by immunohistochemistry and polymerase chain reaction for the evidence of HCMV antigen expression and the presence of viral DNA. We detected HCMV antigen and DNA for IEI-72, pp65, and late antigen in 33/36, 28/36, and 26/36 in glioblastoma multiforme patients whereas 12/14, 10/14, and 9/14 in anaplastic astrocytoma patients, respectively. Furthermore, 84% of glioma patients were positive for immunoglobulin G (IgG) compared to 72.5% among control samples (P = 0.04). These data indicate the presence of the HCMV virus in a high percentage of glioma samples demonstrating distinct histopathological grades and support previous reports showing the presence of HCMV infection in glioma tissue. These studies demonstrate that detection of low-levels of latent viral infections may play an active role in glioma development and pathogenesis.

Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 (“SmyleDCpp65”). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4+ and CD8+ T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34+ cells obtained from PB and CB were transplanted into irradiated NOD.Rag1−/−.IL2γc−/− mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8+ T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4+/CD8+ T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.

The Value of Persistent Cytomegalovirus Infection in the Formation of Groups of Children with Recurrent Acute Respiratory Diseases

So far not defined the value of CMV persistence in frequently and chronically ill children, the impact of active CMV-infection on the severity of the condition and the incidence of viral-bacterial complications. 149 surveyed frequently and long ill children with lymphadenopathy (58), obstructive bronchitis (40), the defeat of the ENT organs (18), meningoencephalitis (13), pleuropneumonia (12), thrombocytopenia (8), were in the offices MDKB. The age of the children ranged from 1 month. up to 14 years. Structural identification of CMV pp65 antigen is recommended in severe patients and in children with frequent and prolonged acute respiratory infections, complications of purulent-inflammatory diseases. To confirm active CMV replication method should be used in real time PCR in saliva and urine (positive at 48.6%).

Cytomegalovirus and Mucoepidermoid carcinoma: A possible causal relationship? A pilot study.

Background:Human cytomegalovirus (CMV) infection has tropism for salivary gland ductal epithelium and establishes a persistent and lifelong infection. Mucoepidermoid carcinoma (MEC) is the most common salivary gland tumor. Recent studies have established that mouse CMV-induced tumorigenesis displays histologic and molecular characteristics similar to human MEC. We wished to explore further down the lane by analyzing the expression of pp65 and the key oncogenic signaling pathway in cases of MEC and their etiological relevance in the Indian scenario as a pilot study.Materials and Methods:Histopathologically confirmed cases of MEC (n = 4) and normal salivary gland tissue (n = 4) were subjected to immunohistochemical analysis using the markers pp65 and epidermal growth factor receptor (EGFR).Results:The pp65 antigen expression was found to be negative in all the studied cases and one case of high-grade MEC showed EGFR expression.Conclusion:The purpose of the study was to explore the role of CMV in the development of MEC, as it might help to exploit this etiological agent as a therapeutic target. Similar to human papillomavirus (HPV), these might identify a subset of neoplasms with a varied biological behavior and alternative therapies. However, this vision is obscured by contradicting evidence in the literature. As of today, surgery remains the only best possible management for these patients unless proven otherwise.

Prospective Analysis of Cytomegalovirus Reactivation and the Immune State of Low-Grade B-Cell Lymphoma Patients Treated with Bendamustine

Bendamustine is an alkylator containing a benzimidazole ring; it only has partial cross-resistance with the other alkylating agents. Several studies have shown that bendamustine is effective as a single agent or as combined treatment with rituximab for low-grade B-cell lymphoma. Although bendamustine has a favorable safety profile, the occurrence of myelosuppression and lymphocytopenia is relatively frequent. In lymphocytopenia, CD4-positive (CD4+) T-cell recovery is specifically impaired. If this occurs over a prolonged period of time, patients are at risk of opportunistic infections, such as cytomegalovirus (CMV) reactivation. This is a risk for bendamustine-treated patients, although the actual frequency of CMV reactivation in these patients is not clear.In this study, we prospectively evaluated the occurrence of CMV reactivation in relapsed or refractory low-grade B-cell lymphoma patients who were treated with bendamustine alone or in combination with rituximab. We simultaneously investigated the immune state of the patients. We analyzed CMV pp65 antigen in leukocytes, immunoglobulin (Ig) G, IgM, and IgA levels, and the CD4+:CD8+ lymphocyte ratio prior to treatment and after the third and sixth courses of treatment.There were 29 patients enrolled in the study. The median age was 68 years old (range, 28–83 years). Patients were treated a median of 2 times prior to bendamustine treatment; these treatments included chemotherapy and radiotherapy. There were 15 patients (51%) who completed 6 courses of chemotherapy.The immune status of the patients is shown in Figure 1. The median CD4+ lymphocyte count prior to treatment was 218/µL, which decreased to 75/µL by the end of treatment. The median of IgG, IgM, and IgA concentrations before treatment were 909, 52, and 147 mg/dL, respectively; by the end of treatment, the IgG, IgM, IgA concentrations had decreased to 832, 30, and 110 mg/dL, respectively. The extent of CD4+ T-cell depression during treatment appeared to be more severe than the Ig depression.CMV pp65 antigen was detected in 3 patients after the third course of treatment, and in 2 patients after the sixth course of treatment. None of the patients had any symptoms and CMV pp65 antigen count returned to negative without any treatment. The median CD4+ lymphocytes count in patients with or without CMV reactivation is shown in Table 1. There is a trend that the depression of CD4+ cells after the 6th course of bendamustine increases the risk of CMV reactivation.This is the first study to prospectively analyze CMV reactivation in low-grade B-cell lymphoma patients treated with bendamustine. In this study, we found that CMV reactivation by bendamustine occurred in approximately 15% of patients. On the other hand, the risk of symptomatic CMV disease, which would require medical intervention, does not appear to be high in bendamustine-treated patients.Most patients with relapsed or refractory low-grade B-cell lymphoma who are administered bendamustine therapy are in an immunosuppressed condition at the beginning of treatment. This is due to heavy chemotherapy pretreatment, which is often performed using rituximab. In addition to this, the decrease in CD4+ lymphocytes observed during bendamustine treatment, is likely to increase the risk of opportunistic infections, such as CMV reactivation. In future studies, we aim to investigate the risk factors for symptomatic cytomegalovirus infection in patients treated with bendamustine, especially in combination with rituximab.We hope that our study, which evaluates the risk of opportunistic infections in bendamustine treated patients, will be beneficial in determining the optimal timing for a preemptive approach for CMV infection. This will be helpful in establishing safe bendamustine treatment regimens in relapsed or refractory low-grade B-cell lymphoma patients. [Display omitted] Table 1The median number of lymphocytes, CD4+ lymphocytes and CD4/8 ratio in patients with or without cytomegalovirus reactivationParameterAll patientsCMV reactivationp value+-Before treatment (n=24)Lymph (/µl)9961518 (n=5)987 (n=19)0.24CD4+ cells (/µl)2182612180.96CD4/8 ratio0.690.600.690.54After the 3rd course (n=23)Lymph (/µl)9801330 (n=5)944 (n=18)0.39CD4+ cells (/µl)1301181300.52CD4/8 ratio0.200.120.200.16After the 6th course (n=15)Lymph (/µl)628579 (n=5)902 (n=10)0.27CD4+ cells (/µl)75361180.035 *CD4/8 ratio0.200.170.210.35*; significant DisclosuresNo relevant conflicts of interest to declare.

Generation of Antigen-Specific Cytotoxic T Lymphocytes with Peripheral B Cells Activated By α-Galactosylceramide

Dendritic cells (DCs) are well known as the most potent professional antigen presenting cells (APCs). Nonetheless, the use of these cells in immunotherapy has been limited due to the time consuming and laborious steps required to generate DCs from monocytes in vitro. Therefore, alternative APCs has drawn much attention because of their relative convenience in manipulation.In this study, the efficacy of B cells as APCs, as compared to DCs, in induction of cytotoxic T lymphocytes (CTLs) against cytomegalovirus (CMV)-specific antigens was evaluated. B cells were isolated by depletion of peripheral blood mononuclear cell (PBMCs) from healthy individuals with MACS system, loaded with α-galactosylceramide (α-GalCer) for inducing B cell activation, and nucleofected with CMV-antigen coding plasmid DNA, pCK-pp65-IRES-IE1. As other APCs, monocyte-derived DCs were induced with various cytokines (GM-CSF, IL-4, IL-1b, TNF-a), for 6 days and nucleofected with the same plasmid DNA. Ag-nucleofected B cells or DCs were cocultured with T cells for 14 days in vitro. The cells were harvested and subsequently immunoassayed.Proliferation of cells was more expanded by about 25~32% in CMV-CTLs induced by DCs compared to of B cells, but there was no significant difference in immunogenicity between CMV-CTLs induced with B cells and DCs. Compared to CMV-CTLs induced by DCs, the CTLs induced by α-GalCer-loaded B cells induced similar cytotoxicity against CMV antigen (Ag) in vitro. The CMV-CTLs by α-GalCer-loaded B cells recognized CMV antigen pp65 (median 88 SFC/105) and IE-1 (median 86 SFC/105) in donor 1, and CMV antigen pp65 (median 31 SFC/105) and IE-1 (median 37 SFC/105) in donor 2. Similarly, the CMV-CTLs by DCs recognized CMV antigen pp65 (median 133 SFC/105) and IE-1 (median 32 SFC/105) in donor 1, and CMV antigen pp65 (median 37 SFC/105) and IE-1 (median 43 SFC/105) in donor 2.Immunogenicities of both CTLs were similar not only on IFN-γ ELISPOT (Enzyme-linked immunospot) assay but also on cytotoxicity assay. The CMV-CTLs by α-GalCer-loaded B cells have killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (85%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (69%, at E:T ratio 10:1) and IE1 (27%, at E:T ratio 10:1) in donor 2. Also, the CMV-CTLs by DCs show killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (42%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (88%, at E:T ratio 10:1) and IE1 (64%, at E:T ratio 10:1) in donor 2.These observations suggest that α-GalCer-loaded B cells could be used in general as APCs instead of DCs. Using the B cells as APCs have several benefits such as cost-effectiveness, less time-consuming, and less laborious compared to when DCs are used. Furthermore, nucleofection technique might be useful in delivering antigen-coding DNA, not only for virus antigens but also for tumor antigens, directly into the nucleus. Our results demonstrate that α-GalCer-loaded B cells could be potent APCs in generating antigen-specific CTLs for cellular vaccines and adoptive immunotherapy.Acknowledgment: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012R1A1A2008316) and we thank Ann M. Leen, Helen E. Heslop and Malcomn K. Brenner from Center for Cell and Gene Therapy, Baylor College of Medicine Center for their kind help. DisclosuresNo relevant conflicts of interest to declare.

IFNγ⁻TNFα⁻IL2⁻MIP1α⁻CD107a⁺PRF1⁺ CD8 pp65-Specific T-Cell Response Is Independently Associated With Time to Death in Elderly Humans.

Persistent cytomegalovirus (CMV) infection has been suggested to be a major driving force in the immune deterioration and an underlying source of age-related diseases in the elderly. CMV antibody titers are associated with lower responses to vaccination, cardiovascular diseases, frailty, and mortality. CMV infection is also associated with shorter T-cell telomeres and replicative senescence. Although an age-related deregulation of CMV-specific T-cell responses could be an underlying cause of the relationship between CMV and immune defects, strong and polyfunctional responses are observed in elderly individuals, casting uncertainty on their direct role in age-related immune frailty. In this study, we longitudinally followed a cohort of healthy donors aged over 50 years, assessing their mortality rates and time to death during a 2-year period. Specific T-cell responses to the immunodominant antigen pp65 (IFNγ, TNFα, IL2, MIP1α, CD107a, and perforin production) were analyzed at the beginning of the 2-year observation period. A cytotoxic CD8 pp65-specific T-cell response, without cytokine or chemokine coexpression, was independently associated with all-cause mortality in these elderly individuals. This pp65-specific CD8 T-cell response could be a useful tool to identify individuals with depressed immune function and a higher risk of death.

HCMV pp65 antigen slides were used to observe the titer of sera anti‐HCMV IgG antibody by indirect fluorescent assay. Apple‐green fluorescence was observed on the slides when sera anti‐HCMV IgG antibodies existed, and the density of fluorescense paralled the concentration of antibodies. Reddish fluorescence was observed if the cells were uninfected by HCMV, and as the background of images.

Microbiology and ImmunologyVolume 58, Issue 8 p. i-i Cover ImageFree Access HCMV pp65 antigen slides were used to observe the titer of sera anti-HCMV IgG antibody by indirect fluorescent assay. Apple-green fluorescence was observed on the slides when sera anti-HCMV IgG antibodies existed, and the density of fluorescense paralled the concentration of antibodies. Reddish fluorescence was observed if the cells were uninfected by HCMV, and as the background of images. Haiyan Xu, Haiyan XuSearch for more papers by this authorPanpan Dong, Panpan DongSearch for more papers by this authorXiaozhou He, Xiaozhou HeSearch for more papers by this authorXuyi Ma, Xuyi MaSearch for more papers by this authorDong Xue, Dong XueSearch for more papers by this authorYanyun Zhang, Yanyun ZhangSearch for more papers by this authorXueguang Zhang, Xueguang ZhangSearch for more papers by this author Haiyan Xu, Haiyan XuSearch for more papers by this authorPanpan Dong, Panpan DongSearch for more papers by this authorXiaozhou He, Xiaozhou HeSearch for more papers by this authorXuyi Ma, Xuyi MaSearch for more papers by this authorDong Xue, Dong XueSearch for more papers by this authorYanyun Zhang, Yanyun ZhangSearch for more papers by this authorXueguang Zhang, Xueguang ZhangSearch for more papers by this author First published: 03 August 2014 https://doi.org/10.1111/1348-0421.12183AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume58, Issue8August 2014Pages i-i RelatedInformation

Highlights of This Issue

Id1 induces activation of PI3K/AKT pathway, but the underlying mechanism remains unknown. In this study, Li and colleagues report that Id1 induces the expression and secretion of IGF2 in several cancer types including esophageal cancer. Id1-induced IGF2 could activate the PI3K/AKT pathway by signaling through IGF1R in an autocrine and endocrine manner to promote tumor progression and metastasis. The study also shows that IGF2 and IGF1R are valid targets in esophageal cancer therapy and that a monoclonal antibody targeting IGF1R (cixutumumab), used alone or combined with cytotoxic chemotherapeutic drugs, has potential clinical applications in systemic treatment of esophageal cancer.Beaver and colleagues demonstrate the ability to detect DNA mutations in plasma from early-stage breast cancer patients. In a prospective study, blood from 29 patients was obtained prior to surgery and used to detect PIK3CA mutations with 93.3% sensitivity and 100% specificity. Five patients had plasma PIK3CA mutations after surgery. This study demonstrates the feasibility of detecting plasma tumor DNA in early-stage breast cancer. Importantly, this provides the foundation for future work examining whether persistent plasma tumor DNA postsurgery could identify patients who may benefit from adjuvant therapies versus those that are already cured after surgery.Follicular lymphomas (FL) establish an inflammatory microenvironment, notably driven by COX-2, with a large amount of PGE2 synthesis. To inhibit the crosstalk between the supporting tumor microenvironment and tumor B-cells, Gallouet and colleagues showed that the use of celecoxib, an anti-inflammatory drug, can reduce in vitro PGE2 production from FL-patients stromal cells. Moreover, the use of delecoxib and TRAIL, a cytotoxic molecule, can sensitize primary B-cells from FL patients to death. This combination of treatments can therefore target both microenvironment and tumor cells for more efficiency in FL treatments.Although detection of low levels of human cytomegalovirus (CMV) expression in glioblastoma (GBM) has been reported by several groups, the physiologic relevance of this expression remains unclear. Nair and colleagues explored whether the CMV antigen pp65 could serve as an effective tumor rejection antigen in primary GBM. Despite deficits in cellular immunity in patients with GBM, CMV pp65-specific T cells could be reliably activated in vitro using pp65 RNA-pulsed autologous dendritic cells. Importantly, CMV-specific T cells were effective in recognizing and killing autologous GBM tumor cells. These studies strongly support the rationale for CMV-targeted immunotherapy in patients with GBM.

Crosstalk of BAFF and TLRs after HCMV Infection in renal transplantation recipients (TRAN3P.878)

Abstract To investigate the correlation of BAFF signal and TLRs activtion after HCMV infection in renal transplant (RTx) recipients. Peripheral blood and urine of RTx recipients (113 cases) were collected. Urine HCMV DNA was detected by quantitative real-time PCR (qPCR); Sera anti-pp65 IgG /IgM antibody were detected with the use of HCMV pp65 antigen slide by indirect immunofluorescence assay; Soluble BAFF (sBAFF) was tested by ELISA; Sera anti-HLA&amp;MICA was determined by single-antigen flow bead assays (One Lambda) on a Luminex platform; The concerned genes selected by Gene Ontology (GO) function classification and KEGG Pathway analysis were tested by qPCR chip assay. In RTx recipients with high copy of urine HCMV DNA (&amp;gt;10,000 copy/ml, 12 cases, positive group), sBAFF, anti-pp65 antibody, and anti-HLA&amp;MICA antibody also increased (P&amp;lt;0.05); The correlation analysis indicated that HCMV DNA level was positively related with sBAFF, anti-pp65 antibody, and anti-HLA&amp;MICA antibody. And the qPCR chip assay results showed that 46 genes were differentially expressed (&amp;gt; 2 times) in all. In positive group BAFF-R and ICAM1 were upregulated, TLR9, NLRC5 and other 20 genes were downregulated, compared with negative group (with low copy of HCMV DNA); IFN-γ and ICAM1 were upregulated and 8 genes were downregulated in positive group, compared with healthy controls. So BAFF signal was enhanced after HCMV infection in RTx recipients, which involved in the decrease of renal allograft function.

Pretransplant CD8 T-Cell Response to IE-1 Discriminates Seropositive Kidney Recipients at Risk of Developing CMV Infection Posttransplant

Cytomegalovirus (CMV) infection is an ongoing clinical problem in solid-organ transplantation (SOT). Pretransplant CMV serology is currently the only tool for assessing the risk of CMV infection, although cellular immune responses driven by CMV-specific CD4 and CD8 T lymphocytes are important for controlling viral replication. Therefore, the analysis of CMV-specific T cells may be useful for estimating the risk of infection. This is a prospective study of patients with kidney transplants and no prophylactic treatment for CMV replication. CD4 and CD8 T-cell responses to the major CMV pp65 and IE-1 antigens in 15 seropositive patients at intermediate risk of CMV infection were investigated, according to current algorithms. Intracellular flow cytometry was employed to determine IFN-γ production as a functional readout. The response was analyzed in pretransplant samples and prospectively at 1 and 6 months and at 1 year posttransplant. It was observed that the CD8 responses to IE-1 antigen were practically absent pretransplant in patients who developed CMV infection posttransplant. Within the group of patients free of infection, CD8 responses to IE-1 were detected more frequently and were significantly higher (P=0.0083). In a receiver operating characteristics curve analysis (AUC=0.929; P=0.010; 95% CI: 0.078-1.0), low CD8 responses to IE-1 (≤0.05%) pretransplant predicted the development of CMV infection under the immunosuppressive regime after transplant with 100% specificity and 85.7% sensitivity. Assessment of IE-1-specific CD8 T-cell frequencies pretransplant may be a useful tool for identifying seropositive SOT patients at risk of developing CMV infection posttransplant.

Low prevalence of active cytomegalovirus infection in a cardiovascular intensive care unit.

Active cytomegalovirus infection is not uncommon in critically ill non-immunosuppressed patients. We conducted a preliminary observational study to determine the prevalence of active cytomegalovirus infection in cardiovascular surgical patients. One hundred patients admitted to the intensive care unit following cardiovascular surgery were enrolled between January 2010 and May 2010. Four patients (4%) were positive for serum pp65 antigens, though cytomegalovirus-positive serology (immunoglobulin G, IgG) was found in 98 patients (98%) including those four patients. Active cardiac diseases and their operative procedures including cardiopulmonary bypass may not be significant risk factors for active cytomegalovirus infection unless systemic derangements are also present.

Heterogeneity of CD4+ and CD8+ T-cell Responses to Cytomegalovirus in HIV-Infected and HIV-Uninfected Men Who Have Sex With Men

Studies of T-cell immunity to human cytomegalovirus (CMV) primarily reflect anti-CMV pp65 or immediate early antigen 1 (IE-1) activity. We assessed responses of T cells from human immunodeficiency virus (HIV)-negative and HIV-infected men to peptide pools spanning 19 CMV open reading frames selected because they previously correlated with total CMV-specific T-cell responses in healthy donors. Cells producing cytokines in response to pp65 or IE-1 together composed <12% and <40% of the total CD4(+) and CD8(+) T-cell responses to CMV, respectively. These proportions were generally similar regardless of HIV serostatus. Thus, analyses of total CMV-specific T-cell responses should extend beyond pp65 and IE-1 regardless of HIV serostatus.

Quiescent Crohn’s Disease in Clinical Remission With 6-Mercaptopurine: A Breeding Ground for CMV Pneumonia as Part of a Viral Infection Domino

Cytomegalovirus (CMV) infection is frequently associated with inflammatory bowel disease (IBD). CMV pneumonitis has been reported infrequently in patients with IBD, while there has been one such report in a patient with quiescent Crohn’s disease (CD) under long-term immunosuppressant treatment with 6-mercaptopurine (6-MP). We present an unusual case of a 24-year-old male, with fistulized CD in clinical remission under 6-MP, who presented with fever and increased seropurulent fistulae effusion. Shortly after admission, he presented nonproductive cough, pancytopenia, elevated serum aminotransferases, hypoxemia and bilateral pulmonary reticulonodular infiltrates expanding from basal bronchopulmonary segments towards the hila. Positive CMV IgM/IgG, pp65 antigen and CMV-PCR confirmed the diagnosis of CMV pneumonitis. Enterococcus faecium and faecalis were also separately isolated from cultures of two different, concurrent enterocutaneous fistulae. Successful treatment included antiviral and appropriate antibiotic therapy. A subsequent adenovirus co-infection in this patient, demonstrating a viral domino phenomenon, illustrates the difficulty of establishing a final diagnosis in a complex case. J Med Cases. 2014;5(2):66-72 doi: http://dx.doi.org/10.14740/jmc1499w