Clotting Changes, Including Disseminated Intravascular Coagulation, during Rapid Renal-Homograft Rejection

One of two patients in whom early homograft rejection developed after renal transplantation had many antidonor antibodies before operation. By the measurement of gradients across intracorporeal and extracorporeal homografts in this patient, the new kidneys were shown to sequester host immunoglobulins, platelets, white cells and clotting factors. Moreover, the renal venous blood then contained fibrinolytic activity. This presensitized recipient, as well as a second patient who did not have detectable preformed humoral antibodies, gave evidence from clinical observation and from the various clotting tests of disseminated intravascular coagulation with fibrinolysis and a severe bleeding diathesis. Immunofluorescent and histologic studies revealed a laying down of fibrin in the homograft vessels that continued in some cases to cortical necrosis of the transplanted kidneys or, alternatively, receded at the time fibrinolysis occurred. The variety of rejection seen in these patients has been characterized as an immunologically induced coagulopathy. © 1970, Massachusetts Medical Society. All rights reserved

Risk of malignant lymphoma associated with human herpesvirus-8: a case–control study in Spain

No overall increased risk of lymphoma associated with antibodies to human herpesvirus-8 was found in 526 lymphomas and 599 controls (odds ratio (OR)=1.04, 95% confidence interval (CI)=0.62-1.75); significant increases were noted for 19 lymphoplasmacytic lymphomas (OR=4.47, 95% CI=1.34-14.85) and nine low-grade lymphoma/lymphoma B-cell NOS (OR=5.82, 95% CI=1.07-31.73)

Mutational analysis of the latency-associated nuclear antigen DNA-binding domain of Kaposi's sarcoma-associated herpesvirus reveals structural conservation among gammaherpesvirus origin-binding proteins

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus functions as an origin-binding protein (OBP) and transcriptional regulator. LANA binds the terminal repeats via the C-terminal DNA-binding domain (DBD) to support latent DNA replication. To date, the structure of LANA has not been solved. Sequence alignments among OBPs of gammaherpesviruses have revealed that the C terminus of LANA is structurally related to EBNA1, the OBP of Epstein–Barr virus. Based on secondary structure predictions for LANADBD and published structures of EBNA1DBD, this study used bioinformatics tools to model a putative structure for LANADBD bound to DNA. To validate the predicted model, 38 mutants targeting the most conserved motifs, namely three α-helices and a conserved proline loop, were constructed and functionally tested. In agreement with data for EBNA1, residues in helices 1 and 2 mainly contributed to sequence-specific DNA binding and replication activity, whilst mutations in helix 3 affected replication activity and multimer formation. Additionally, several mutants were isolated with discordant phenotypes, which may aid further studies into LANA function. In summary, these data suggest that the secondary and tertiary structures of LANA and EBNA1 DBDs are conserved and are critical for (i) sequence-specific DNA binding, (ii) multimer formation, (iii) LANA-dependent transcriptional repression, and (iv) DNA replication

Bim Nuclear Translocation and Inactivation by Viral Interferon Regulatory Factor

Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8) uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1–4), which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFβ receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control replication-induced apoptosis and suggest that inhibitory targeting of vIRF-1:Bim interaction may provide an effective antiviral strategy

Non-Human Primate Model of Kaposi's Sarcoma-Associated Herpesvirus Infection

Since Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) was first identified in Kaposi's sarcoma (KS) lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA) were readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells

Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material

HOX-mediated LMO2 expression in embryonic mesoderm is recapitulated in acute leukaemias

The Lim Domain Only 2 (LMO2) leukaemia oncogene encodes an LIM domain transcriptional cofactor required for early haematopoiesis. During embryogenesis, LMO2 is also expressed in developing tail and limb buds, an expression pattern we now show to be recapitulated in transgenic mice by an enhancer in LMO2 intron 4. Limb bud expression depended on a cluster of HOX binding sites, while posterior tail expression required the HOX sites and two E-boxes. Given the importance of both LMO2 and HOX genes in acute leukaemias, we further demonstrated that the regulatory hierarchy of HOX control of LMO2 is activated in leukaemia mouse models as well as in patient samples. Moreover, Lmo2 knock-down impaired the growth of leukaemic cells, and high LMO2 expression at diagnosis correlated with poor survival in cytogenetically normal AML patients. Taken together, these results establish a regulatory hierarchy of HOX control of LMO2 in normal development, which can be resurrected during leukaemia development. Redeployment of embryonic regulatory hierarchies in an aberrant context is likely to be relevant in human pathologies beyond the specific example of ectopic activation of LMO2

Reactive Oxygen Species Hydrogen Peroxide Mediates Kaposi's Sarcoma-Associated Herpesvirus Reactivation from Latency

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the host following an acute infection. Reactivation from latency contributes to the development of KSHV-induced malignancies, which include Kaposi's sarcoma (KS), the most common cancer in untreated AIDS patients, primary effusion lymphoma and multicentric Castleman's disease. However, the physiological cues that trigger KSHV reactivation remain unclear. Here, we show that the reactive oxygen species (ROS) hydrogen peroxide (H2O2) induces KSHV reactivation from latency through both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic replication, and KSHV reactivation from latency induced by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines are mediated by H2O2. Mechanistically, H2O2 induction of KSHV reactivation depends on the activation of mitogen-activated protein kinase ERK1/2, JNK, and p38 pathways. Significantly, H2O2 scavengers N-acetyl-L-cysteine (NAC), catalase and glutathione inhibit KSHV lytic replication in culture. In a mouse model of KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and significantly prolongs the lifespan of the mice. These results directly relate KSHV reactivation to oxidative stress and inflammation, which are physiological hallmarks of KS patients. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs could be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies

K13 blocks KSHV lytic replication and deregulates vIL6 nad hIL6 expression: A model of lytic replication induced clonal selection in viral oncogenesis

Background. Accumulating evidence suggests that dysregulated expression of lytic genes plays an important role in KSHV (Kaposi's sarcoma associated herpesvirus) tumorigenesis. However, the molecular events leading to the dysregulation of KSHV lytic gene expression program are incompletely understood. Methodoloxy/Principal Findings. We have studied the effect of KSHV-encoded latent protein vFLIP K13, a potent activator of the NF-κB pathway, on lytic reactivation of the virus. We demonstrate that K13 antagonizes RTA, the KSHV lytic-regulator, and effectively blocks the expression of lytic proteins, production of infectious virions and death of the infected cells. Induction of lytic replication selects for clones with increased K13 expression and NF-κB activity, while siRNA-mediated silencing of K13 induces the expression of lytic genes. However, the suppressive effect of K13 on RTA-induced lytic genes is not uniform and it falls to block RTA-induced viral IL6 secretion and cooperates with RTA to enhance cellular IL-6 production, thereby dysregulating the lytic gene expression program. Conclusions/Significance. Our results support a model in which ongoing KSHV, lytic replication selects for clones with progressively higher levels of K13 expression and NF-κB activity, which in turn drive KSHV tumorigenesis by not only directly stimulating cellular survival and proliferation, but also indirectly by dysregulating the viral lytic gene program and allowing non-lytic production of growth-promoting viral and cellular genes. Lytic Replication-Induced Clonal Selection (LyRICS) may represent a general mechanism in viral oncogenesis. 2007 Zhao et al