HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer.

The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi's sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.

Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination.

A Luminex (Luminex Corp., Austin, TX) multiplex swine cytokine assay was developed to measure 8 cytokines simultaneously in pig serum for use in assessment of vaccine candidates. The fluorescent microsphere immunoassay (FMIA) was tested on archived sera in a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine/challenge study. This FMIA simultaneously detects innate (IL-1 beta, IL-8, IFN-alpha, TNF-alpha, IL-12), regulatory (IL-10), Th1 (IFN-gamma) and Th2 (IL-4) cytokines. These proteins were measured to evaluate serum cytokine levels associated with vaccination strategies that provided for different levels of protective immunity against PRRSV. Pigs were vaccinated with a modified-live virus (MLV) vaccine and subsequently challenged with a non-identical PRRSV isolate (93% identity in the glycoprotein (GP5) gene). Protection (as defined by no serum viremia) was observed in the MLV vaccinated pigs after PRRSV challenge but not those vaccinated with killed virus vaccine with adjuvant (KV/ADJ) (99% identity in the GP5 gene to the challenge strain) or non-vaccinates. Significantly elevated levels of IL-12 were observed in the KV/ADJ group compared to MLV vaccinated and control groups. However, this significant increase in serum IL-12 did not correlate with protection against PRRSV viremia. Additional studies using this assay to measure the local cytokine tissue responses may help in defining a protective cytokine response and would be useful for the targeted design of efficacious vaccines, not only for PRRSV, but also for other swine pathogens.

Antibody response to porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural proteins and implications for diagnostic detection and differentiation of PRRSV types I and II.

To further characterize the humoral immune response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV), direct enzyme-linked immunosorbent assays (ELISA) were used to study the kinetics of antibody responses directed against PRRSV nonstructural proteins in pigs experimentally exposed to the virus. The highest immunoreactivities were against nsp1, nsp2, and nsp7. Using the recombinant nsp7 as an antigen, we validated a dual ELISA for the simultaneous detection and differentiation of serum antibodies against type I and type II PRRSV. Receiver operating characteristic analysis based on 1,334 known-positive and 1,357 known-negative samples showed good specificity (98.3% to type I and 99.3% to type II) and sensitivity (97.4% for type I and 99.8% for type II). To differentiate type I and type II PRRSV, 470 sera originating from experimentally inoculated pigs were tested, and positive sera were correctly differentiated in 469 of 470 samples. The capability of the nsp7 dual ELISA to detect serum antibody responses from pigs infected with various genetically different field strains was determined. The nsp7 dual ELISA possessed 97.6% agreement with the Idexx HerdChek PRRS 2XR ELISA. In further testing of Idexx ELISA suspected false-positive samples, the nsp7 dual ELISA resolved 98% of the samples as negative. Taken together, these results indicate that the nsp7 dual ELISA can be used as a differential test for PRRSV serology with high levels of sensitivity and specificity. This ELISA offers an additional tool for routine or follow-up diagnostics, as well as having substantial value in epidemiological surveys and outbreak investigations.

Cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid.

Porcine reproductive and respiratory virus (PRRSV) is an enveloped positive-sense RNA virus of the family Arteriviridae that causes severe and persistent disease in pigs worldwide. The PRRSV virion consists of a lipid envelope that contains several envelope proteins surrounding a nucleocapsid core that encapsidates the RNA genome. To provide a better understanding of the structure and assembly of PRRSV, we have carried out cryo-electron microscopy and tomographic reconstruction of virions grown in MARC-145 cells. The virions are pleomorphic, round to egg-shaped particles with an average diameter of 58 nm. The particles display a smooth outer surface with only a few protruding features, presumably corresponding to the envelope protein complexes. The virions contain a double-layered, hollow core with an average diameter of 39 nm, which is separated from the envelope by a 2-3 nm gap. Analysis of the three-dimensional structure suggests that the core is composed of a double-layered chain of nucleocapsid proteins bundled into a hollow ball.

Monocyte enrichment of mononuclear apheresis preparations with a multistep back-flush procedure on a cord blood filter.

INTRODUCTION: Monocytes or mononuclear cells have been investigated for the treatment of chronic wounds and spinal cord injuries, as well as serve as a source for dendritic or endothelial cell culture. Because these cells may have clinical benefit yet no rapid and inexpensive closed system for monocyte purification is commercially available, a method was investigated to enrich monocytes from mononuclear apheresis units using a cord blood filter. METHODS: A 4-step method for monocyte enrichment was developed which involved 1) filtering a mononuclear apheresis unit through a cord blood filter, 2) chasing with medium to remove non-adherent residual cells and plasma, 3) back-flushing under low shear conditions to remove loosely adherent lymphocytes, and 4) back-flushing under high shear conditions to collect a fraction enriched in monocytes. Apheresis units and enriched monocyte preparations were characterized by cell count and differential, filter-isolated preparations were cryopreserved, and thawed preparations were assayed for viability, and phagocytosis. Enriched monocyte preparations were also assayed for inflammatory cytokines secretion and secretion of prostaglandin E2 during short-term culture. RESULTS: Monocytes were viable, capable of phagocytosis, and enriched using the multistep filter elution technique to represent 42 +/- 13-percent of white cells in the final preparation. Fifty-three-percent of monocytes were recovered in the final preparation, while total cell counts of red cells, platelets, neutrophils and lymphocytes were reduced to 3.0, 3.0, 4.5 and 16-percent, respectively, from levels present in mononuclear apheresis units. Filter enriched monocyte preparations secreted IL-8, IL-6, MCP-1, and MIP-1alpha, during short term culture. CONCLUSION: The use of a multi-step back flush procedure with a cord blood filter resulted in rapid enrichment of viable and functional monocytes from mononuclear apheresis units with significant reduction of contaminating platelets and red cells.

Dendritic cell culture: a simple closed culture system using ficoll, monocytes, and a table-top centrifuge.

Dendritic cells (DCs) are potent antigen-presenting cells involved in the induction of T cell-mediated immune responses and as such have emerged as important candidates for cellular-based therapies. Critical to safe clinical use is the easy manipulation of DCs and their precursors in a closed system. We have developed a serum-free, closed culture system applying a simple wash-Ficoll centrifugation method to reduce platelet and red blood cell (RBC) contamination. This procedure optimized adherence of monocytes (44 +/- 10.9% recovery, >85% expressed CD14(+)/CD163(+)) for the generation of DCs from mononuclear cell (MNC) apheresis units. Most RBCs and up to 98% of platelets were removed. Following density sedimentation, cell viability remained high (98 +/- 2%) with only minimal loss of monocytes (3 +/- 3%). Importantly, Ficoll-treated monocytes retained their ability to differentiate to mature DCs demonstrated by morphology, phenotype (MHC class II(+), CD1a(+), CD80(+), CD86(+), and CD83(+)), ability to stimulate mixed lymphocyte responses (MLR), present antigen, and produce interleukin-12 (IL-12). Nonadherent CD3(+) (80 +/- 4%) were also isolated for functional assays. Ficoll can be easily incorporated into a simple adherence-based closed system for collection of lymphocytes and adherent monocytes for DC culture. The procedure is relatively fast (effective working time 5-6 h), does not impair monocyte function or induce substantial cell activation, and can be performed economically using equipment found in a typical blood banking environment.

A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment.

BACKGROUND: Cryopreservation and storage permitting multiple treatments with single donations is of practical importance to cellular therapies. HES and DMSO, used successfully in simple clinical procedures for freezing marrow and peripheral blood progenitor cells at -80 degrees C, was tested on antigen-presenting dendritic cells (DCs) and cells used in their derivation. STUDY DESIGN AND METHODS: DCs cultured in serum-free media from adherent or CD14+ apheresis MNCs (n = 36) in the presence of GM-CSF + IL4 +/- TNFalpha were frozen and stored at -80 degrees C in 6-percent HES, 5-percent DMSO, and 4-percent HSA. Apheresis MNCs, CD14+ monocytes, and lymphocytes were similarly frozen and later thawed for culture. Cells were assayed for viability, DC phenotype, mixed lymphocyte reaction, and antigen presentation before and 3, 6, 9, 12 or more months after freezing. RESULTS: DCs retained viability (82 +/- 2.3%) for at least 24 months. Mature and immature phenotype and function were preserved. Thawed MNCs and CD14+ cells differentiated to DCs and lymphocytes maintained high functional viability (92 +/- 3%) comparable to prefreeze levels. CONCLUSION: A simple -80 degrees C freezing and storage method that combines extracellular (HES) and intracellular (DMSO) agents is practical and preserves functional viability of DCs, MNCs, CD14+ monocytes, and lymphocytes.