Codon Deoptimization of UL54 in meq-Deleted Marek's Disease Vaccine Candidate Eliminates Lymphoid Atrophy but Reduces Vaccinal Protection.

Marek's disease (MD) is an oncogenic, lymphoproliferative, and highly contagious disease of chickens. Its etiologic agent is the alphaherpesvirus Marek's disease virus (MDV, Gallid alphaherpesvirus 2), and it is a chronic and ubiquitous problem for the poultry industry with significant economic impact in the United States and worldwide. We have previously demonstrated that MDV attenuated by dicodon deoptimization of the UL54 gene results in reduced gene product accumulation in vitro, with reduced viral genome copy number upon infection and reduced atrophy of bursa and thymus in vivo as well. In this report we detail our attempts to use the same attenuation strategy on a meq-deleted MDV mutant, rMd5B40ΔMeq. Unlike the wild-type rMd5B40 virus the rMd5B40ΔMeq is no longer oncogenic, but infected birds experience an unacceptable amount of bursa and thymus atrophy (BTA). We produced two meq-deleted MDV recombinants with a dicodon-deoptimized UL54 (rMd5B40ΔMeq/UL54deop1 and -deop2) and tested their tendency to cause BTA and to serve as a protective vaccine. We found that, although dicodon deoptimization of the UL54 gene results in a virus that spares the infected animal from atrophy of the bursa and thymus, the meq-deleted UL54-deoptimized recombinant is also less protective than the meq-deleted virus without UL54 deoptimization, the HVT + SB1 combination vaccine, or the Rispens (CVI988) vaccine.

Attenuation of a recombinant Marek's disease virus lacking the meq oncogene and evaluation on its immune efficacy against Marek's disease virus.

SC9-2 is a recombinant Marek's disease virus (MDV) strain lacking the meq oncogene. Previous study demonstrated that SC9-2 virus provides good protection against challenge with a very virulent MDV rMd5, but it induces immunosuppressive effects in specific pathogen-free (SPF) chickens. In the present study, SC9-2 was serially passaged on chicken embryo fibroblast (CEF) cell cultures. The pathogenicity and immune efficacy of SC9-2/10th and SC9-2/40th against rMd5 were evaluated. Animal experimental results showed that SC9-2/10th and SC9-2/40th showed no lethality or tumorigenicity in SPF chickens. Body weight of chickens inoculated with SC9-2/40th were significantly higher than that of the chickens inoculated with SC9-2/10th but lower than that of the uninoculated controls. The severity of bursa and thymus atrophy (BTA) and spleen enlargement in SC9-2/40th-inoculated chickens were also weaker than the SC9-2/10th-inoculated ones but stronger than the uninoculated controls. Chickens inoculated with SC9-2/40th and SC9-2/10th showed similar antibody levels induced by H9N2 subtype avian influenza virus/Newcastle disease virus inactivated vaccines, both of which were lower than the uninoculated controls. Replication of SC9-2/40th was significantly lower than SC9-2/10th in feather follicle epithelium (FFE) of infected chickens. The immune protection index of SC9-2/40th was also lower than that of SC9-2/10th, but the difference was not significantly, and both of which were significant higher than that of the commercial MDV vaccine CVI988/Rispens. The results of our studies demonstrated that SC9-2/40th showed weaker severity of BTA, spleen enlargement, and body weight loss and lower replication level in FFE than SC9-2/10th in SPF chickens. However, SC9-2/40th was able to confer better immune protection as compared with CVI988/Rispens vaccination in SPF chickens. In conclusion, serially attenuation of SC9-2 in CEFs reduced the lymphoid organ atrophy and replication in SPF chickens, and the immune protective efficacy of attenuated viruses was still superior than CVI988/Rispens.

The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells.

In the present research, we assumed that reducing the amounts of E6 and E7 oncoproteins by a specific siRNA sequence and recovering p53 and RB proteins, along with the recovery of the FOXO1 protein by applying anti-miR-182, would increase apoptosis and reduce proliferation rate in cancer cells. The HPV16-positive CaSki cervical cancer cell line was used. 48 hours after transfection of siRNA for targeting E6 and E7 oncoproteins and anti-miR-182, expression of its cellular targets p53, p21 and FOXO1 was assessed by real-time PCR, western blot analysis and immunocytofluorescence staining. In all treatments, apoptosis rate and viability were evaluated using Annexin-V-FITC apoptosis detection kits and MTT assays, respectively. Among the designed siRNAs, E6-1 and E7-2 proved the most effective in reducing E6 and E7 expressions by increasing the apoptotic rates to 12.4% and 16%, respectively, after 48 hours. Also, using anti-miR-182 increased apoptotic rate to 12.7% 48 hours after transfection of cervical cancer cells. The combinational use of either E6-1 or E7-2 siRNAs with anti-miR-182 resulted in a rise in apoptosis to 19.3% and 26%, respectively, higher than those obtained from the individual application of either without anti-miR-182. The simultaneous use of siRNA E6-1 and siRNA E7-2 with cisplatin increased sensitivity to cisplatin and reduced the viability of the cancer cells as compared to the use of cisplatin alone. The simultaneous use of cisplatin and anti-miR-182 had no considerable effect on viability or apoptosis rate compared to cisplatin alone.

Evidence of disrupted high-risk human papillomavirus DNA in morphologically normal cervices of older women.

High-risk human papillomavirus (HR-HPV) causes nearly 100% of cervical carcinoma. However, it remains unclear whether HPV can establish a latent infection, one which may be responsible for the second peak in incidence of cervical carcinoma seen in older women. Therefore, using Ventana in situ hybridisation (ISH), quantitative PCR assays and biomarkers of productive and transforming viral infection, we set out to provide the first robust estimate of the prevalence and characteristics of HPV genomes in FFPE tissue from the cervices of 99 women undergoing hysterectomy for reasons unrelated to epithelial abnormality. Our ISH assay detected HR-HPV in 42% of our study population. The majority of ISH positive samples also tested HPV16 positive using sensitive PCR based assays and were more likely to have a history of preceding cytological abnormality. Analysis of subsets of this population revealed HR-HPV to be transcriptionally inactive as there was no evidence of a productive or transforming infection. Critically, the E2 gene was always disrupted in those HPV16 positive cases which were assessed. These findings point to a reservoir of transcriptionally silent, disrupted HPV16 DNA in morphologically normal cervices, re-expression of which could explain the increase in incidence of cervical cancer observed in later life.

Anthracene-9, 10-dione derivatives induced apoptosis in human cervical cancer cell line (CaSki) by interfering with HPV E6 expression.

A new series of anthracene-9, 10-dione derivatives have been synthesized to increase cytotoxic activity against human papillomavirus (HPV) positive cancer cell line, CaSki. The highest cytotoxicity was achieved by 4-(benzylamino)-9,10-dioxo-4a,9,9a,10-tetrahydroanthracen-1-yl 4-ethylbenzenesulfonate (5) with the inhibitory concentration 50 (IC50) of 0.3 µM which is 20 times lower than that of cisplatin (CDDP; IC50 = 8.0 µM). The toxicity against non-cancerous cell line, WI-38, was low with the IC50 > 10 µM. Treatment with this compound resulted in decreasing HPV E6 expression. Furthermore, increasing p53 and decreasing Bcl-2 expression were noted. Cell cycle profiles revealed an accumulation of cells in the G2/M phase.

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis.

In vitro studies have implicated the Lyn tyrosine kinase in erythropoietin signaling. In this study, we show that J2E erythroid cells lacking Lyn have impaired signaling and reduced levels of transcription factors STAT5a, EKLF and GATA-1. Since mice lacking STAT5, EKLF or GATA-1 have red cell abnormalities, this study also examined the erythroid compartment of Lyn(-/-) mice. Significantly, STAT5, EKLF and GATA-1 levels were appreciably lower in Lyn(-/-) erythroblasts, and the phenotype of Lyn(-/-) animals was remarkably similar to GATA-1(low) animals. Although young adult Lyn-deficient mice had normal hematocrits, older mice developed anemia. Grossly enlarged erythroblasts and florid erythrophagocytosis were detected in the bone marrow of mice lacking Lyn. Markedly elevated erythroid progenitors and precursor levels were observed in the spleens, but not bone marrow, of Lyn(-/-) animals indicating that extramedullary erythropoiesis was occurring. These data indicate that Lyn(-/-) mice display extramedullary stress erythropoiesis to compensate for intrinsic and extrinsic erythroid defects.

Reduced dosage of Bruton's tyrosine kinase uncouples B cell hyperresponsiveness from autoimmunity in lyn-/- mice.

The development of autoimmunity is correlated with heightened sensitivity of B cells to B cell Ag receptor (BCR) cross-linking. BCR signals are down-regulated by Lyn, which phosphorylates inhibitory receptors. lyn(-/-) mice have reduced BCR signaling thresholds and develop autoantibodies, glomerulonephritis, splenomegaly due to myeloid hyperplasia, and increased B-1 cell numbers. Bruton's tyrosine kinase (Btk), a critical component of BCR signaling pathways, is required for autoantibody production in lyn(-/-) mice. It is unclear whether Btk mediates autoimmunity at the level of BCR signal transduction or B cell development, given that lyn(-/-)Btk(-/-) mice have a severe reduction in conventional B and B-1 cell numbers. To address this issue, we crossed a transgene expressing a low dosage of Btk (Btk(low)) in B cells to lyn(-/-)Btk(-/-) mice. Conventional B cell populations were restored to levels similar to those in lyn(-/-) mice. These cells were as hypersensitive to BCR cross-linking as lyn(-/-) B cells as measured by proliferation, Ca(2+) flux, and activation of extracellular signal-regulated kinase and Akt. However, lyn(-/-)Btk(low) mice did not produce anti-ssDNA, anti-dsDNA, anti-histone, or anti-histone/DNA IgM or IgG. They also lacked B-1 cells and did not exhibit splenomegaly. Thus, B cell hyperresponsiveness is insufficient for autoimmunity in lyn(-/-) mice. These studies implicate B-1 and/or myeloid cells as key contributors to the lyn(-/-) autoimmune phenotype.

The Lyn tyrosine kinase negatively regulates neutrophil integrin signaling.

The Src family kinase Lyn has been shown to play both stimulatory and inhibitory roles within several hemopoietic cell types. In this study, we investigated the role played by Lyn in neutrophil integrin signaling. Loss of Lyn resulted in a hyperresponsive phenotype on engagement of surface integrins at low valency. Lyn(-/-) neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when adherent to surfaces coated with either cellular counterreceptors or extracellular matrix proteins. In contrast, Lyn-deficient and wild-type cells expressed similar levels of surface integrins and responded equivalently to activating agents in suspension, indicating that the enhanced responses of lyn(-/-) cells was specific to the integrin signaling pathways. Lyn-deficient macrophages also displayed a hyperadhesive phenotype. Biochemical analysis of macrophages from lyn(-/-) mice revealed that Lyn plays an essential role in the adhesion-dependent phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of the inhibitory receptors SIRP1alpha and PIR-B, which in turn recruit the phosphatase SHP-1. These observations suggest that reduced mobilization of SHP-1 to the membrane in lyn(-/-) neutrophils results in a hyperadhesive and hyperactive phenotype. This hypothesis is further supported by the fact that neutrophils from me(v)/me(v) mice, which have significantly reduced SHP-1 activity, are also hyperresponsive following integrin engagement. This is the first direct evidence using primary leukocytes from lyn(-/-) mice that this kinase functions as a negative regulator in integrin signaling.