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Necrotic enteritis (NE) is a common disease that causes great economic loss to the broiler industry due to mortality and reduced performance. Although Clostridium perfringens (CP) is a necessary component of this disease, coccidia species are a well-defined predisposing factor that exacerbates the condition. Different Eimeria species have been reported to influence NE to different degrees. In a pair of experiments, six different Eimeria species were evaluated in the presence and absence of C. perfringens . Male broiler chicks were housed in battery cages for the duration of both experiments. Feed conversion, body weight gain, and NE mortality were reported in both experiments. Experiment 1 challenged birds with E. maxima , E. acervulina , E. tenella , E. necatrix , and E. brunetti at day 13 and subsequently inoculated birds with CP on days 18, 19, and 20. In the second experiment, E. maxima , E. acervulina , E. tenella , and E. praecox were inoculated on day 15 and challenged with CP on days 17, 18, 19, 20, 21, and 22 of the experiment. In the first experiment, E. acervulina , E. brunetti , E. maxima , and E. necatrix with the addition of CP all stimulated necrotic enteritis mortality. In the second experiment, E. praecox had minimal impact on performance during the challenge (14-23 days) while E. maxima + CP decreased body weight gain and increased mortality compared to the CP alone control. Eimeria maxima had the highest mortality (21.9%) in this experiment followed by E. acervulina (6.3%). The remaining Eimeria with added CP in the second experiment did not induce NE mortality. While the challenge with CP alone did not induce mortality, feed conversion was increased compared to the unchallenged control group. When using isolated Eimeria species in these experiments, disturbances created by E. brunetti and E. maxima resulted in the most-severe challenges. These experiments highlight the NE risk of these species of Eimeria and give insight into how other species interact with the host in a controlled CP challenge model.

Aims: To design and synthesize novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluate their analgesic and anti-inflammatory potential., Main Methods: The murine macrophage cell line RAW 264.7 has been widely used as a model for inflammatory responses in vitro. Our model consists of cultured monolayers of RAW 264.7 cells in which media concentrations of 15-deoxy-Δ(13,14)-PGJ2 (PGJ) are measured by ELISA following LPS (10ng/ml) stimulation and treatment with 0.1, 0.3, 1.0, 3.0 and 10μM concentrations of the compounds., Key Findings: Our data indicate that several of our compounds have the capacity to increase production of PGJ and may also increase the occurrence of programmed cell death (apoptosis)., Significance: Thus these agents are potential candidates for the therapy of conditions characterized by ongoing (chronic) inflammation and its associated pain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Several N-linked amino acid-linoleic acid conjugates were studied for their potential as anti inflammatory agents. The parent molecule, N-linoleoylglycine was tested in an in vivo model, the mouse peritonitis assay where it showed activity in reducing leukocyte migration at doses as low as 0.3mg/kg when administered by mouth in safflower oil. Harvested peritoneal cells produced elevated levels of the inflammation-resolving eicosanoid 15-deoxy-Δ(13,14)-PGJ(2). These results are similar to those obtained in earlier studies with N-arachidonoylglycine. An in vitro model using mouse macrophage RAW cells was used to evaluate a small group of structural analogs for their ability to stimulate 15-deoxy-Δ(13,14)-PGJ(2) production. The d-alanine derivative was the most active while the d-phenylalanine showed almost no response. A high degree of stereo specificity was observed comparing the d and l alanine isomers; the latter being the less active. It was concluded that linoleic acid conjugates could provide suitable templates in a drug discovery program leading to novel agents for promoting the resolution of chronic inflammation., (Copyright © 2011. Published by Elsevier Ltd.)

The expression of oncostatin M and leukemia inhibitory factor (LIF), JAK-STAT activators and members of the interleukin-6 family of cytokines, were examined in a series of primary ovarian carcinomas using immunohistochemistry. The malignant epithelial cells of all 29 ovarian carcinomas examined expressed oncostatin M; none expressed LIF. Oncostatin M can activate two related receptors, one consisting of a low-affinity LIF receptor subunit, LIFR beta, which forms a heterocomplex with the gp130 signal transducing protein and can recognize both oncostatin M and LIF, and a second heterocomplex consisting of a subunit that specifically recognizes oncostatin M, OSMR beta, and the gp130 protein. By immunohistochemistry, 25 of 25 ovarian carcinomas examined expressed the LIFR beta subunit in the malignant epithelial cells (all samples express gp130), and two-thirds the ovarian carcinomas studied expressed OSMR beta mRNA as determined by RT-PCR. Thus oncostatin M and its receptors are commonly coexpressed in malignant ovarian epithelial cells, and represent a potential autocrine loop in this tumor type. STAT3, of one the signaling proteins downstream of the oncostatin M/LIF receptors, was found in its phosphorylated, activated form (phosphotyrosine 705 STAT3) in the malignant epithelial cells of 17 of 23 ovarian carcinomas examined (74%) as determined by immunohistochemistry; this suggests that this protein is constitutively activated in most ovarian carcinomas, as it is in many other human malignancies. Recombinant human Oncostatin M (rhOSM) can induce the transient tyrosine 705 phosphorylation of STAT3 in serum-starved LIFR beta/OSMR beta expressing ovarian carcinoma cell lines, but does not alter cell growth and effects only a modest increase in the apoptotic rate in these cultured cells. Oncostatin M and its receptors may be part of a network of cytokine systems within ovarian carcinomas that may act to maintain STAT3 in its activated form, a phenomenon associated with the malignant phenotype., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

Immunohistochemistry was used to determine the expression of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in primary ovarian carcinomas. The expression of G-CSFR was observed in the malignant cells of each of the 46 primary carcinomas examined; G-CSF was coexpressed in both the malignant epithelial cells and the stroma of 56.5% of the specimens. Thus the majority of ovarian carcinomas harbor both potential autocrine and paracrine G-CSF axes. In 37% of the samples, G-CSF was expressed only within stromal cells, suggesting that only a potential paracrine system is in place. In a preliminary, retrospective, evaluation, the survival of patients whose tumors expressed only the apparent paracrine loop was significantly worse than patients whose tumors expressed both potential autocrine and paracrine G-CSF-based regulatory loops (14.5 vs. 42.5 months, respectively). Studies on the potential function of G-CSF were performed using the G-CSFR-expressing OVCAR-3 ovarian carcinoma line. As a single agent, rhG-CSF failed to stimulate [3H]-thymidine incorporation in these cells, but enhanced the mitogenic action of epidermal growth factor (EGF) in a dose-dependent manner. Thus, potential autocrine and/or paracrine loops involving G-CSF and its receptor occur in over 90% of primary ovarian carcinomas, and may act to modulate the action of growth factors.

The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus (EBV) is selectively expressed in the Reed-Sternberg (RS) cells of EBV-associated Hodgkin's disease (HD). However, no differences in clinical presentation and course are found between EBV positive and EBV negative forms of HD suggesting a common pathogenetic mechanism. We have studied the LMP1 associated signaling pathways and their dominant negative inhibition in the myelomonocytic HD-MyZ and the B-lymphoid L-428 HD cell lines. In both EBV negative cell lines expression of LMP1 is associated with the formation of multinuclear RS cells. Dominant negative inhibition of NF-kappa B mediated signaling at the step of I kappa B-alpha phosphorylation results in increased cell death with only a few typical RS cells resistant to overexpression of the dominant negative inhibitor I kappa B-alpha-N delta 54. However, dominant negative inhibition of NF-kappa B mediated signaling at the early step of TRAF2 interaction results in the formation of multinuclear cells in both cell lines and, in addition, in clusters of small mononuclear cells in the HD-MyZ cell line. In HD-MyZ cells overexpression of the powerful JBD-inhibitor of the JNK signal transduction pathway is restricted to small cells and never observed in RS cells. These small cells undergo apoptosis as shown by the TUNEL technique. Apoptosis of small cells is still observed after co-transfection of JBD and LMP1 but in addition a few apoptotic HD-MyZ cells with large fused nuclear masses are identified suggesting that specific inhibition of JNK leads also to apoptosis of LMP1 induced RS cells. Thus, activation of the JNK signaling pathway is also important in the formation of Reed-Sternberg cells. Our findings are consistent with a model where all three LMP1 associated functions, i.e. NF-kappa B mediated transcription, TRAF2 dependent signaling, and c-Jun activation act as a common pathogenetic denominator of both EBV negative and EBV positive HD.

We report the case of a 30-year-old woman who presented with an EBV related hemophagocytic syndrome. After a few months she developed a T-cell rich B-cell non-Hodgkin's lymphoma with liver involvement. Serological data demonstrated a reactivation of the EBV infection. Tumor progression with liver involvement occurred during treatment with conventional chemotherapy. Tumor reduction and disappearance of all masses was seen after starting high-dose sequential chemotherapy, followed by an autologous peripheral blood progenitor transplantation LMP-1 could be amplified in the tumor material by PCR technology, but no LMP-1 expression could be found in the few malignant B-cells with Reed-Sternberg morphology. Sequence analysis of the carboxy terminal of the LMP-1 region revealed the naturally occurring 30 bp deletion variant of the LMP-1 with multiple point mutations within the NF kb region. Since LMP-1 was not expressed in the malignant tumor cells, no evidence could be found, that EBV participated in the tumorigenesis of this case.

EBNA-2 is the first protein to be detected after infection of primary B lymphocytes by Epstein-Barr virus (EBV) and plays an essential role as transcriptional activator in EBV-induced lymphocyte transformation. We analysed by PCR and sequencing regions of the EBNA-2 type 1 gene from isolates from 13 children with infectious mononucleosis (IM), 6 children with tonsillar hyperplasia (TH), and 9 patients with HIV infection followed longitudinally. We found in all three groups of patients frequent non-silent point mutations at positions 48990, 48991, 49021, 49057, 49083, 49089, 49091, 49113, 49119, 49140, 49156, and a triplet insertion at position 49136. While 4 out of 13 samples from patients with IM showed a mosaic pattern suggesting co-existence of more than 1 substrain of EBNA-2 type 1, none of the samples from TH showed this pattern consistent with substrain selection during clinical latency. No sequence changes were noted over time in samples derived from patients with HIV infection. We conclude that in analogy to the coexistence of several subtypes of EBNA-1 in healthy EBV carriers, samples from IM can harbor more than one subtype of the EBNA-2 type 1 gene.

To assess the frequency of malignancy-associated 30-bp deletion variants of the latent membrane protein 1 (LMP-1) in benign conditions, a comparative sequence analysis was done using samples from 20 American children with acute infectious mononucleosis and 16 Swiss children with chronic tonsillar hyperplasia. The 30-bp deletion variant (LMP-1-del) was present in 66% of patients (12/20 with infectious mononucleosis and 12/16 with tonsillar hyperplasia). Two additional patients had a 3-bp deletion and an inframe insertion of 18 nucleotides, respectively. All but 1 isolate had numerous nonsilent point mutations. These data identify a hypervariable region within the C-terminus of LMP-1, in a domain required for maximal stimulation of NF-kappaB activity. These data demonstrate that LMP-1-del variants are frequent in acute infectious mononucleosis and tonsillar hyperplasia and identical to those observed in Epstein-Barr virus-associated AIDS-related lymphoma.

The latent membrane protein 1 (LMP1) oncogene is one of the major proteins synthesized by the Epstein-Barr virus (EBV) and is expressed in Reed-Sternberg (RS) cells of EBV-associated Hodgkin's disease (HD). We have studied the effect of this oncoprotein on the formation of RS cells in the EBV-negative HD cell lines L-428 and KM-H2 as well as on the formation of multinucleated cells in the mononuclear human embryonic kidney cell line 293. LMP1 prototype (B95-8) and its naturally occurring carboxy terminal 30 base pair deletion variant LMP1-del were transfected into the cell lines and cytocentrifuge preparations were analysed after 24, 48, 72, 144, 216, and 240 h. While no oncoprotein expression was seen in the KM-H2 cell lines, expression of LMP1 and LMP1-del was observed in the L-428 and 293 cell lines. In the HD cell line L-428 oncoprotein expression was infrequent but when observed was very strong and preferentially associated with multinucleated RS cell morphology (71% of LMP1 positive cells). This is in contrast with the untransfected or transfected but not expressing cells where intermediate mononuclear elements predominated over multinucleated RS cells (< 3%). Frequent oncoprotein expression was observed in the 293 cells and again was associated with multinuclearity. These LMP1 expressing 293 giant cells showed strong expression of ICAM-1(CD54), not detectable in the untransfected cells. In the LMP1-del transfectants giant cells with more than four nuclei were frequently observed. However, giant cells were much less frequent in 293 cells transfected with the amino terminal deletion variant of LMP1 or the lytic form of LMP1, known to induce low NF-kappa B activation compared to the LMP1 prototype. Therefore, LMP1 mediated NF-kappa B activation appears to be involved in polycaria formation. The strong association of LMP1 expression with multinuclearity in a genetically unstable condition -the L-428 and 293 cells show multiple chromosomal abnormalities-suggests that this oncoprotein including its naturally occurring carboxy terminal deletion variant promote the formation of multinuclear cells, in particular of RS cells in EBV-associated HD.

A 74-year-old woman developed angioimmunoblastic lymphadenopathy (AILD) with involvement of intra-abdominal and retroperitoneal lymph nodes. Southern blot analysis showed germline configuration of the JH genes and an oligoclonal pattern of the TcR beta genes. The immunoblasts were of B-cell phenotype and often expressed the CD30 antigen and the latent membrane protein 1 (LMP1) oncogene. Six nonsilent point mutations were identified near the 3' end of the LMP1 gene, leading to a cluster of six amino acid changes within a protein domain needed for maximal NF-kappa B stimulation. After a clinical remission of 8 months the patient relapsed with generalized lymphadenopathy and died secondary to tuberculosis. The oligoclonal rearrangements of the TcR beta genes may reflect an unsuccessful cellular immune response to Mycobacterium tuberculosis or an HLA-restricted T-cell response to B-immunoblasts expressing mutated viral antigens. A positive percutaneous tuberculin test observed 6 months prior to the onset of AILD is in favor of the first possibility.

This sequencing study of 17 acquired immunodeficiency syndrome-related lymphomas (9 primary brain, 8 systemic) and 8 human immunodeficiency virus-negative atypical lymphoproliferations expressing large amounts of the latent membrane protein 1 (LMP1) of Epstein-Barr virus was performed to characterize the carboxy terminal NF-kappa B activation domain of LMP1 at the molecular level in an immunocompromised host. In-frame deletions within the NF-kappa B activation domain were identified in all but 2 primary brain lymphomas, 4 systemic lymphomas, and 4 atypical lymphoproliferations, ie, in 60% of cases. In addition, non silent point mutations (range 1 to 5, mean 3.3) were detected in all cases. Although all changes occurred within the first 100 nucleotides of the carboxy terminal NF-kappa B activation domain--a critical sequence for the protein half-life--not a single point mutation was found in the remaining 62 nucleotides, necessary for malignant transformation. Such a clustering of nonrandom sequence variations, associated with a high oncoprotein expression in immunocompromised hosts, suggests that this part of the LMP1 oncogene behaves as a hypervariable region with natural selection of growth-promoting variants through prolongation of the protein half-life.

Nonconservative mutations were introduced by site-specific mutagenesis into the fusion peptide and the adjacent heptad repeat region of the fusion protein of Newcastle disease virus in order to determine the role of both regions in the fusion activity of the protein. Mutations in both regions that allowed for proper folding and intracellular transport of the protein blocked the fusion activity of the protein when assayed in the presence of the hemagglutinin-neuraminidase protein.

Phenylalanine is the amino acid at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains of Newcastle disease virus, which have a leucine residue in this position (Toyoda et al. (1989) Virology 169, 273-282). To explore the role of this phenylalanine in the fusion activity of the protein, this residue, amino acid 117 in the fusion protein sequence, was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. While both wild-type and the F117G protein were proteolytically cleaved and F1 was detected, the F117L protein was not cleaved. In the presence of the HN protein, both wild-type F and F117G proteins stimulated fusion, but the F117L protein was inactive in fusion. However, incubation in trypsin activated the fusion activity of the protein. Thus the phenylalanine at the amino terminus of the F1 component of the fusion protein is not required for the fusion activity of the protein. The presence of a leucine at this position blocks cleavage even though the cleavage site sequence is unchanged.

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion.

The hemagglutinin-neuraminidase (HN) gene and the phosphoprotein (P) gene of Newcastle disease virus (NDV) were inserted into a replication competent avian leukosis virus vector. The expression of the HN gene from this vector in chick embryo cells has been previously reported. The P gene is also expressed from this vector in chick embryo cells. The retroviruses were used to immunize 4-week-old chickens. Birds receiving the virus containing the HN gene developed low levels of serum HI titers and NDV neutralization titers. Upon challenge, all birds vaccinated with the HN gene containing virus were protected from disease but not viral infection and replication. In contrast, birds immunized with the P gene containing retrovirus developed more severe clinical signs of disease earlier than birds receiving no immunization or retrovirus alone. The results obtained with the HN gene may have potential application to reducing disease due to NDV genetically engineered vaccines.

Characterization of the posttranslational modifications of the mature, cell-associated hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) revealed that the HN protein exists in two forms differentiated by disulfide bonds and glycosylation. One form, HNa, contains intermolecular disulfide bonds and is endoglycosidase H partially resistant. The other form, HNb, is not linked by disulfide bonds and is endoglycosidase H sensitive. Both forms of the protein are modified with fucose indicating transport to the Golgi membranes. Both forms are detected at the cell surface by monoclonal antibody. Furthermore, both forms are transported to the cell surface with identical kinetics. HNa is incorporated into virions. HNb is not incorporated into virions and is presumably degraded. The cDNA derived from the HN gene was expressed from a retrovirus vector. The majority of the protein expressed was in the nonvirion-associated form b. Evidence is presented that the level of gene expression determines the ratio of the two forms of HN protein. At high levels of expression, the virion-associated form is favored while at low levels of expression the nonvirion-associated form is favored. The results presented have implications for persistent infections as well as expression of viral genes from different vectors.

We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M andG proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations.

The nucleotide sequence of cloned cDNA copies of the mRNA encoding the Newcastle disease virus (NDV), strain AV, phosphoprotein (P) was determined. The sequence of 1443 nucleotides contains one long open reading frame which could encode a protein with a molecular weight of 42,126, and two smaller open reading frames which could encode proteins with molecular weights of 11,178 and 13,935. Full-length cDNA clones were constructed in an SP6 vector, mRNA was transcribed in a cell-free system using the SP6 polymerase, and the mRNA was translated in a wheat germ cell-free extract. The P mRNA directed the synthesis of, primarily, four products. One, with a molecular weight of 53,000 Da, comigrated with authentic P protein made in infected cells and was precipitable with antisera with specificity for the NDV P protein. The other products of the cell-free reaction had molecular weights of 38,000, 29,000 and 12,000. The 29,000- and the 38,000-Da polypeptides were also precipitable with anti-P protein antibody. Using truncated cDNA clones, evidence is presented that the 38,000- and 29,000-Da proteins are derived from initiation at AUG triplets in the same reading frame as the P protein. Infected cells also contain these polypeptides which may be analogous to C proteins of other paramyxoviruses. Thus the NDV P protein mRNA is different than most other paramyxovirus P protein mRNAs which are translated in two different reading frames to yield the P and C proteins.

Prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and salicylic acid were measured in blood samples from 10 healthy men after administration of antiinflammatory doses of aspirin (3.9 g/day) or salsalate (3.0 g/day). Each medication was given for 3 days, followed by an observation period of 13 days. Plasma salicylate concentrations were slightly, but generally insignificantly, higher during aspirin dosing, although both drugs produced salicylic acid levels in the antiinflammatory range. Serum levels of PGE2 and TXB2, which reflected synthesis of cyclo-oxygenase products by platelets, were minimally affected by salsalate but profoundly suppressed by aspirin. When medication was discontinued, the effects of salsalate on serum PGE2 and TXB2 were readily reversible within 36 h, whereas the recovery from aspirin was still incomplete after 13 days of observation. These results indicate that the two orally administered salicylates have differential effects on prostaglandin synthesis in platelets and may also differ in their therapeutic and adverse effects.