Prion protein amyloidosis with divergent phenotype associated with two novel nonsense mutations in PRNP

Stop codon mutations in the gene encoding the prion protein (PRNP) are very rare and have thus far only been described in two patients with prion protein cerebral amyloid angiopathy (PrP-CAA). In this report, we describe the clinical, histopathological and pathological prion protein (PrPSc) characteristics of two Dutch patients carrying novel adjacent stop codon mutations in the C-terminal part of PRNP, resulting in either case in hereditary prion protein amyloidoses, but with strikingly different clinicopathological phenotypes. The patient with the shortest disease duration (27 months) carried a Y226X mutation and showed PrP-CAA without any neurofibrillary lesions, whereas the patient with the longest disease duration (72 months) had a Q227X mutation and showed an unusual Gerstmann-Sträussler-Scheinker disease phenotype with numerous cerebral multicentric amyloid plaques and severe neurofibrillary lesions without PrP-CAA. Western blot analysis in the patient with the Q227X mutation demonstrated the presence of a 7 kDa unglycosylated PrPSc fragment truncated at both the N- and C-terminal ends. Our observations expand the spectrum of clinicopathological phenotypes associated with PRNP mutations and show that a single tyrosine residue difference in the PrP C-terminus may significantly affect the site of amyloid deposition and the overall phenotypic expression of the prion disease. Furthermore, it confirms that the absence of the glycosylphosphatidylinositol anchor in PrP predisposes to amyloid plaque formation

Transcriptional Analysis Implicates Endoplasmic Reticulum Stress in Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. To date, the disease process is still poorly understood. In this study, brain tissue samples from animals naturally infected with BSE were analysed to identify differentially regulated genes using Affymetrix GeneChip Bovine Genome Arrays. A total of 230 genes were shown to be differentially regulated and many of these genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response and transcription. Seventeen genes are associated with the endoplasmic reticulum (ER) and 10 of these 17 genes are involved in stress related responses including ER chaperones, Grp94 and Grp170. Western blotting analysis showed that another ER chaperone, Grp78, was up-regulated in BSE. Up-regulation of these three chaperones strongly suggests the presence of ER stress and the activation of the unfolded protein response (UPR) in BSE. The occurrence of ER stress was also supported by changes in gene expression for cytosolic proteins, such as the chaperone pair of Hsp70 and DnaJ. Many genes associated with the ubiquitin-proteasome pathway and the autophagy-lysosome system were differentially regulated, indicating that both pathways might be activated in response to ER stress. A model is presented to explain the mechanisms of prion neurotoxicity using these ER stress related responses. Clustering analysis showed that the differently regulated genes found from the naturally infected BSE cases could be used to predict the infectious status of the samples experimentally infected with BSE from the previous study and vice versa. Proof-of-principle gene expression biomarkers were found to represent BSE using 10 genes with 94% sensitivity and 87% specificity

Isolation of Hox Cluster Genes from Insects Reveals an Accelerated Sequence Evolution Rate

Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution

Resistance to the CCR5 Inhibitor 5P12-RANTES Requires a Difficult Evolution from CCR5 to CXCR4 Coreceptor Use

Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16–18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a “resistant” variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching

Antibody Responses against Xenotropic Murine Leukemia Virus-Related Virus Envelope in a Murine Model

Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV.Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations.Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans

Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3), an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to beta- and gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6) mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (Δ5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the Δ5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function

A prospective study of nomogram-based adaptation of prostate radiotherapy target volumes

Background: A prospective clinical trial was conducted to evaluate the feasibility of a novel approach to the treatment of patients with high risk prostate cancer (HRPC) through the use of a nomogram to tailor radiotherapy target volumes. Methods: Twenty seven subjects with HRPC were treated with a mildly hypofractionated radiotherapy regimen using image-guided IMRT technique between Jun/2013-Jan/2015. A set of validated prognostic factors were inputted into the Memorial-Sloan-Kettering Cancer Center (MSKCC) prostate cancer nomogram to estimate risk of loco-regional spread (LRS). The nomogram risk estimates for extra-capsular extension (ECE), seminal vesicles involvement (SVI), and pelvic lymph nodes involvement (LNI) were used to adapt radiotherapy treatment volumes based on a risk threshold of ≥15% in all cases. A planning guide was used to delineate target volumes and organs at risk (OAR). Up to three dose levels were administered over 28 fractions; 70Gy for gross disease in the prostate +/- seminal vesicles (2.5Gy/fraction), 61.6Gy for subclinical peri-prostatic disease (2.2Gy/fraction) and 50.4Gy to pelvic nodes (1.8Gy/fraction). Data regarding protocol adherence, nomogram use, radiotherapy dose distribution, and acute toxicity were collected. Results: Nomogram use: 100% of patients were treated for ECE, 88.9% for SVI, and 70.4% for LNI. The three areas at risk of LRS were appropriately treated according to the study protocol in 98.8% cases. The MSKCC nomogram estimates for LRS differed significantly between the time of recruitment and analysis. Contouring protocol compliance: Compliance with the trial contouring protocol for up to seven target volumes was 93.0% (159/171). Compliance with protocol for small bowel contouring was poor (59.3%). Dose constraints compliance: Compliance with dose constraints for target volumes was 97.4% (191/196). Compliance with dose constraints for OAR was 88.2% (285/323). Acute toxicity: There were no grade 3 acute toxicities observed. 20/27 (74.1%) and 6/27 (22.2%) patients experienced a grade 2 genitourinary and gastrointestinal toxicity respectively. Conclusions: We have demonstrated the feasibility of this novel risk-adapted radiation treatment protocol for HRPC. This study has identified key learning points regarding this approach, including the importance of standardization and updating of risk quantification tools, and the utility of an observer to verify their correct use