scholarly journals Quantitative Analysis of Latent Human Cytomegalovirus

1999 ◽  
Vol 73 (6) ◽  
pp. 4806-4812 ◽  
Author(s):  
Barry Slobedman ◽  
Edward S. Mocarski
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Copy Number ◽  
Mononuclear Cells ◽  
Latent Infection ◽  
Natural Infection ◽  
Viral Genomes ◽  
Reverse Transcription Pcr ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of viral DNA was investigated by PCR-driven in situ hybridization (PCR-ISH), and the number of viral genomes per cell was estimated by quantitative competitive PCR during both experimental and natural latent infection. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in >90% of cells at a copy number of 1 to 8 viral genomes per cell. During natural infection, viral genomes were detected in 0.004 to 0.01% of mononuclear cells from granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow from seropositive donors, at a copy number of 2 to 13 genomes per infected cell. When evaluated by reverse transcription–PCR-ISH, only a small proportion of experimentally infected cells (approximately 2%) had detectable latent transcripts. This investigation identifies the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode currently identified latent transcripts.

scholarly journals Latently KSHV-Infected Cells Promote Further Establishment of Latency upon Superinfection with KSHV

2021 ◽  
Vol 22 (21) ◽  
pp. 11994
Author(s):  
Chen Gam ze Letova ◽  
Inna Kalt ◽  
Meir Shamay ◽  
Ronit Sarid
Keyword(s):  
Latent Infection ◽  
Fluorescent Proteins ◽  
Cell Types ◽  
Lytic Cycle ◽  
Viral Reservoir ◽  
Virus Spread ◽  
Viral Genomes ◽  
Latently Infected ◽  
Infected Cells

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related virus which engages in two forms of infection: latent and lytic. Latent infection allows the virus to establish long-term persistent infection, whereas the lytic cycle is needed for the maintenance of the viral reservoir and for virus spread. By using recombinant KSHV viruses encoding mNeonGreen and mCherry fluorescent proteins, we show that various cell types that are latently-infected with KSHV can be superinfected, and that the new incoming viruses establish latent infection. Moreover, we show that latency establishment is enhanced in superinfected cells compared to primary infected ones. Further analysis revealed that cells that ectopically express the major latency protein of KSHV, LANA-1, prior to and during infection exhibit enhanced establishment of latency, but not cells expressing LANA-1 fragments. This observation supports the notion that the expression level of LANA-1 following infection determines the efficiency of latency establishment and avoids loss of viral genomes. These findings imply that a host can be infected with more than a single viral genome and that superinfection may support the maintenance of long-term latency.

scholarly journals Large number of rebounding/founder HIV variants emerge from multifocal infection in lymphatic tissues after treatment interruption

2015 ◽  
Vol 112 (10) ◽  
pp. E1126-E1134 ◽  
Author(s):  
Meghan K. Rothenberger ◽  
Brandon F. Keele ◽  
Stephen W. Wietgrefe ◽  
Courtney V. Fletcher ◽  
Gregory J. Beilman ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Single Gene ◽  
Virus Production ◽  
Lymphoid Tissues ◽  
Anatomic Site ◽  
Peripheral Blood Mononuclear ◽  
Latently Infected ◽  
Infected Cells ◽  
Recrudescent Infection

Antiretroviral therapy (ART) suppresses HIV replication in most individuals but cannot eradicate latently infected cells established before ART was initiated. Thus, infection rebounds when treatment is interrupted by reactivation of virus production from this reservoir. Currently, one or a few latently infected resting memory CD4 T cells are thought be the principal source of recrudescent infection, but this estimate is based on peripheral blood rather than lymphoid tissues (LTs), the principal sites of virus production and persistence before initiating ART. We, therefore, examined lymph node (LN) and gut-associated lymphoid tissue (GALT) biopsies from fully suppressed subjects, interrupted therapy, monitored plasma viral load (pVL), and repeated biopsies on 12 individuals as soon as pVL became detectable. Isolated HIV RNA-positive (vRNA+) cells were detected by in situ hybridization in LTs obtained before interruption in several patients. After interruption, multiple foci of vRNA+ cells were detected in 6 of 12 individuals as soon as pVL was measureable and in some subjects, in more than one anatomic site. Minimal estimates of the number of rebounding/founder (R/F) variants were determined by single-gene amplification and sequencing of viral RNA or DNA from peripheral blood mononuclear cells and plasma obtained at or just before viral recrudescence. Sequence analysis revealed a large number of R/F viruses representing recrudescent viremia from multiple sources. Together, these findings are consistent with the origins of recrudescent infection by reactivation from many latently infected cells at multiple sites. The inferred large pool of cells and sites to rekindle recrudescent infection highlights the challenges in eradicating HIV.

Abstract 239: Stem Cell Therapy Improves Critical Limb Ischemia

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Alaa Marzouk
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Embryonic Stem ◽  
Limb Ischemia ◽  
Control Group ◽  
Chronic Limb Ischemia

Introduction: The journey from single cell to complex being is attributable to stem cells role. Adult stem cells originate during ontogeny & persist in specialized niches within organs. Asymmetric division of each stem cell during differentiation produces : one daughter stem cell & one daughter transit amplifying/intermediate cell having migratory properties. Forced migration of hematopoietic stem/progenitor cells (HSPC) from bone marrow into peripheral blood is called mobilization. Accumulating evidence suggests that attenuation of the chemokine stromal derived factor-1(SDF-1)-CXCR4 axis that plays a pivotal role in retention of HSPC in bone marrow (BM) results in the release of these cells from the BM into peripheral blood. Recently, adult cells have been genetically reprogrammed to an embryonic stem cell like state. Induced pluripotent stem cells (IPSCs) were similar to human embryonic stem cells in morphology, proliferative capacity, expression of cell surface antigens, & gene expression. Treatment of ischemic vascular disease of lower limbs remains a significant challenge. Unfortunately, if medical & surgical salvage procedures fail, amputation is an unavoidable result for those patients. Aim of Work: (Hypothesis) To assess the application of implantation of autologous stem/progenitor cell in the treatment of chronic limb ischemia & to evaluate the safety, efficacy & feasibility of this novel therapeutic approach. Methods: A total of 24 patients with chronic limb ischemia not eligible for arterial reconstruction or endovascular procedures were enrolled & randomized (1:1) to either the implanted group or the control group. Control group: Conventional medical therapy in the form of anti platelet therapy & vasodilators. Implanted group: Subcutaneous injection of 300μ g/day of recombinant human granulocyte colony stimulating factor (G-CSF) for 5 days to mobilize stem/progenitor cells from BM. Total leucocytic count is measured daily to follow up successful mobilization of bone marrow mononuclear cells (BMMNCs). Stem cell Harvesting After 5 days peripheral blood mononuclear cells (PBMNCs) were harvested using a cell separator. Samples from apheresis products are subjected to TLC measurement & immunophenotypic characterization of CD34+ cells by flow cytometry. The collected PBMNCs were implanted by multiple intramuscular injections into ischemic limbs. Results: There was significant increase in pain free walking distance & ankle/brachial index (ABI) & significant decreased rest pain. Effectiveness was documented by : reduced number of amputation, increase ABI & improvement of the quality of life in therapeutic group compared to control group. Conclusion: The novel therapeutic approach of PBMNCs implantation in patients with chronic limb ischemia is safe, feasible & effective in decreasing co-morbidity & rate of amputation. Safety was manifested by absence of complications during G-CSF therapy or during harvesting & injection of the stem cells. Recommendations: 1- Future studies on larger number of patients & longer follow up. 2- Controlled studies using different methods & different cell population (PBMNCs, BMMNCs or MSCs) to compare the outcome of each. 3-Studing the role of endothelial progenitor cell dysfunction in different ischemic diseases to develop successful gene therapy.

scholarly journals Atypical hodgkin and reed-sternberg cells in peripheral blood of a patient with advanced stage of Hodgkin's disease - a case report

2003 ◽  
Vol 131 (9-10) ◽  
pp. 400-402 ◽  
Author(s):  
Rajko Milosevic ◽  
Milica Colovic ◽  
Vesna Cemerikic-Martinovic ◽  
Natasa Colovic ◽  
Marina Bogunovic
Keyword(s):  
Bone Marrow ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Hodgkin's Disease ◽  
Mononuclear Cells ◽  
Hodgkin’S Disease ◽  
Lymphoid Cells ◽  
High Dose ◽  
Advanced Stage ◽  
Neck Lymph Nodes

The occurrence of abnormal Hodgkin's and Reed-Sternberg cells in the peripheral blood in a patient suffering from Hodgkin's disease has been noticed exceptionally rare in a previous period, and especially rare in last ten years primarily due to successfull treatment of this disease. The presence of atypical mononuclear cells in peripheral blood which cytomorphologically resembled Reed-Sternberg cells was registered in 8 patients till 1966. During the last decade, the presence of atypical mononuclear cells in the peripheral blood was used for their isolation cultivation, and detailed immunophenotypic and genetic analysis. The analysis of mononuclear cells in rare patients with Hodgkin's disease was established that they belong to the B-lymphoid cells with expression of CD30 and CD15 antigens. The examination of presence of Hodgkin's cells in the peripheral blood of patients with Hodgkin's disease is important for patients with advanced stage of the disease in which autologous stem cell transplantation and high dose chmeotherapy is planned. The authors present a 33-year-old patient, who noticed enlarged neck lymph nodes in September 2000, high temperature and loss in weight. On physical examination enlarged neck lymph nodes 5x8 cm and hepatosplenomegaly were found. There was anemia and thrombo-cytopenia, and normal WBC count with 24% of lymphoid elements in differential formula. On histologic examination of lymph nodes Hodgkin?s disease, type nodular sclerosis with mixed cellularity was found. Histology of bone marrow showed nodal lymphomatous infiltration. Immunohistochemistry with monoclonal antibodies of concentrate of peripheral blood cells showed expression of CD30+ and CD15+, immunophenotypically and morphologically matching Reed-Sternberg cells. Cytogentic analysis of mononuclear cells of the bone marrow showed normal karyotype. The patient was in clinical stage IV/V of the disease and chemotherapy with 9 cycles of ABVD+Mp protocol was applied. He is still in remission.

Peripheral-blood or bone-marrow mononuclear cells for therapeutic angiogenesis?

The Lancet ◽  
2002 ◽  
Vol 360 (9350) ◽  
pp. 2083 ◽  
Author(s):  
Shoichi Inaba ◽  
Kensuke Egashira ◽  
Kimihiro Komori
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Therapeutic Angiogenesis ◽  
Bone Marrow Mononuclear Cells

Abstract 4535: Therapeutic Angiogenesis by Mononuclear Cell Implantation for Critical Digit Ischemia of Patients with Connective Tissue Diseases

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Masafumi Takahashi ◽  
Yoshiaki Ishigatsubo ◽  
Kazuteru Fujimoto ◽  
Masaaki Miyamoto ◽  
Seiji Minota ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Connective Tissue ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Connective Tissue Diseases ◽  
Clinical Manifestations ◽  
Therapeutic Angiogenesis ◽  
Diffuse Fibrosis ◽  
Dramatic Improvement ◽  
Skin Ulcers

Systemic sclerosis (SSc or scleroderma) is an autoimmune connective tissue disease characterized by diffuse fibrosis, degenerative changes, and microvascular abnormalities. The vasculopathy mainly affects small arteries and capillaries and causes insufficient blood flow, which leads to clinical manifestations, such as Raynaud’s syndrome, fingertip ulcers, and gangrene. Recently, implantation of bone marrow-derived mononuclear cells (MNCs) has been successfully used for therapeutic neovascularization in Buerger’s disease that is thought to be an “autoimmune” vasculitis. The purpose of this study is to determine the efficacy of autologous MNC implantation into the ischemic digits of patients with connective tissue diseases. This study was performed as a prospective, non-randamized, and multicenter clinical trial. Thirty-four patients (19 SSc, 5 SLE, 2 CREST, 2 MCTD, 4 APS, 2 PN) who had painful ischemic digits with skin ulcers were enrolled in this study. Autologous MNCs obtained from bone marrow or peripheral blood were implanted into the ischemic digits. Ischemic pain and ulcers improved remarkably after MNC implantation. In particular, SSc patients showed dramatic improvement of these parameters (18 of 19 patients, 94.7%). In patients with other types of connective tissue diseases, pain and ulcers improved in 12 of 15 patients (80.0%). No serious adverse event was observed. These results demonstrate that implantation of autologous MNCs from bone marrow or peripheral blood into ischemic digits is feasible, safe and effective for improvement of pain and skin ulcers in patients with connective tissue diseases including SSc. Thus, larger, randomized and controlled trials for this cell therapy in patients with connective tissue diseases will be warranted.

Abstract 697: Mobilization of Oct-4 + Bone Marrow-Derived Very Small Embryonic-Like Stem Cells in Patients with Acute Myocardial Infarction

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Wojciech Wojakowski ◽  
Magda Kucia ◽  
Boguslaw Machalinski ◽  
Edyta Paczkowska ◽  
Joanna Ciosek ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Stem Cells ◽  
Bone Marrow ◽  
Pluripotent Stem Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Small Cells ◽  
Acute Mi

Bone marrow-derived CD34 + CXCR4 + progenitor cells are mobilized into peripheral blood early in acute myocardial infarction (MI). Adult murine bone marrow contains population of small CD34 + lin − CD45 − CXCR4 + cells expressing markers of pluripotent stem cells (PSC) SSEA, Oct-4 and Nanog. This population of very small embryonic-like cells (VSEL) has unique morphology (small size 2– 4 μm, large nucleus, euchromatin) and capability to form embrioid bodies (EB). Murine EB-derived cells can in vitro differentiate into cells from all three germ layers including cardiomyocytes. We hypothesized that in patients with acute MI small cells expressing the VSEL immunophenotype and PSC markers are present in bone marrow and mobilized into peripheral blood. Blood samples (20 mL) from 18 patients with acute MI were obtained after 12 hours, 2 and 5 days after symptoms onset. Bone marrow samples (20 mL) were obtained from 2 patients with acute MI and 3 healthy volunteers. Mononuclear cells were isolated using hypotonic lysis and samples were analyzed by FACS. Mobilization of following cell populations was confirmed: hematopoietic lin − CD45 + CXCR4 + , lin − CD45 + CD133 + , lin − CD45 + CD34 + and non-hematopoietic (VSEL) lin − CD45 − CXCR4 + , lin − CD45 − CD133 + , lin − CD45 − CD34 + . Analysis of the cell number using lymphocyte gate showed more significant increase of CD45 + (hematopoietic) populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells. After gating for small events (VSEL size range) we found more significant mobilization of small, non-hematopoietic populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells (Table ). The expression of PSC markers (Oct-4, Nanog, SSEA-1) in VSEL was confirmed using real-time RT-PCR. Conclusion: We report for the first time that acute MI is associated with mobilization of non-hematopoietic VSELs expressing pluripotent stem cells markers.

scholarly journals Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Angela Tsai ◽  
Alivelu Irrinki ◽  
Jasmine Kaur ◽  
Tomas Cihlar ◽  
George Kukolj ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Antiviral Immunity ◽  
Type I ◽  
Toll Like Receptor ◽  
Peripheral Blood Mononuclear ◽  
Effector Functions ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv

ABSTRACT Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs. IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently maintained in reservoirs of latently infected cells. Consequently, lifelong therapy is required to maintain viral suppression. Ultimately, new therapies that specifically target and eliminate the latent HIV reservoir are needed. Toll-like receptor agonists are potent enhancers of innate antiviral immunity that can also improve the adaptive immune response. Here, we show that a highly selective TLR7 agonist, GS-9620, activated HIV from peripheral blood mononuclear cells isolated from HIV-infected individuals with suppressed infection. GS-9620 also improved immune effector functions that specifically targeted HIV-infected cells. Previously published studies on the compound in other chronic viral infections show that it can effectively induce immune activation at safe and tolerable clinical doses. Together, the results of these studies suggest that GS-9620 may be useful for treating HIV-infected individuals on suppressive antiretroviral therapy.

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