Development of an automated human scent olfactometer and its use to evaluate detection dog perception of human scent.

Working Dogs have shown an extraordinary ability to utilize olfaction for victim recovery efforts. Although instrumental analysis has chemically characterized odor volatiles from various human biospecimens, it remains unclear what perceptually constitutes human scent (HS) for dogs. This may be in part due to the lack of methodology and equipment to train and evaluate HS perception. The aims of this research were 1) to develop an automated human scent olfactometer (AHSO) to present HS to dogs in a controlled setting and 2) use the AHSO to evaluate dogs' response to different scented articles and individual components of HS. A human volunteer was placed in a clear acrylic chamber and using a vacuum pump and computer-controlled valves, the headspace of this chamber was carried to one of three ports in a different room. Dogs were trained to search all three ports of the olfactometer and alert to the one containing HS. In Experiment 1 and 2, the AHSO was validated by testing two dogs naïve to HS (Experiment 1) and five certified Search and Rescue (SAR) teams naïve to the apparatus (Experiment 2). All dogs showed sensitivity and specificity to HS > 95% in the apparatus. In Experiment 3, we used a spontaneous generalization paradigm to evaluate generalization from the HS chamber to different scented articles exposed to the same volunteer and to a breath sample. Dogs' response rate to the different scented articles was < 10% but exceeded 40% for the breath sample. In Experiment 4, we replicated this result by re-testing spontaneous generalization to breath and when the volunteer had breath exhausted/removed from the chamber. Dogs' response rate to breath alone was 88% and only 50% when breath was removed. Altogether, the data indicate that exhaled breath is an important and salient component of HS under these conditions.

Real-world evidence of obinutuzumab and venetoclax in previously treated patients with chronic lymphocytic leukemia or small lymphocytic lymphoma.

Venetoclax-obinutuzumab (Ven-O) is frequently administered off-label in relapsed/refractory (r/r) CLL/SLL where venetoclax-rituximab is the approved regimen. We conducted this retrospective, real-world study to evaluate Ven-O in r/r CLL/SLL. Between 7/2019 and 6/2022, 40 patients with r/r CLL/SLL on Ven-O were included. The median age was 72, 28.2% had TP53 mutation and/or 17p deletion, median number of prior therapies was 1 (range, 1-6), and 55% had prior BTK inhibitor exposure. The overall response rate was 90% (complete response [CR] or CR with incomplete marrow recovery in 27.5% and partial response in 62.5%) of patients, and the 2-year progression-free survival was 81.2% (95% CI, 69.5-94.8). Therapy was well tolerated. No laboratory or clinical TLS occurred with venetoclax (Howard criteria). One (3%) patient experienced laboratory TLS with obinutuzumab initiation. In summary, this retrospective cohort study demonstrated that Ven-O achieves frequent, durable responses and can be safely administered in r/r CLL/SLL.

Tumor-resident Lactobacillus iners confer chemoradiation resistance through lactate-induced metabolic rewiring.

Tumor microbiota can produce active metabolites that affect cancer and immune cell signaling, metabolism, and proliferation. Here, we explore tumor and gut microbiome features that affect chemoradiation response in patients with cervical cancer using a combined approach of deep microbiome sequencing, targeted bacterial culture, and in vitro assays. We identify that an obligate L-lactate-producing lactic acid bacterium found in tumors, Lactobacillus iners, is associated with decreased survival in patients, induces chemotherapy and radiation resistance in cervical cancer cells, and leads to metabolic rewiring, or alterations in multiple metabolic pathways, in tumors. Genomically similar L-lactate-producing lactic acid bacteria commensal to other body sites are also significantly associated with survival in colorectal, lung, head and neck, and skin cancers. Our findings demonstrate that lactic acid bacteria in the tumor microenvironment can alter tumor metabolism and lactate signaling pathways, causing therapeutic resistance. Lactic acid bacteria could be promising therapeutic targets across cancer types.

Immune environment and antigen specificity of the T cell receptor repertoire of malignant ascites in ovarian cancer.

We evaluated the association of disease outcome with T cell immune-related characteristics and T cell receptor (TCR) repertoire in malignant ascites from patients with high-grade epithelial ovarian cancer. Ascitic fluid samples were collected from 47 high-grade epithelial ovarian cancer patients and analyzed using flow cytometry and TCR sequencing to characterize the complementarity determining region 3 TCR ß-chain. TCR functions were analyzed using the McPAS-TCR and VDJ databases. TCR clustering was implemented using Grouping of Lymphocyte Interactions by Paratope Hotspots software. Patients with poor prognosis had ascites characterized by an increased ratio of CD8+ T cells to regulatory T cells, which correlated with an increased productive frequency of the top 100 clones and decreased productive entropy. TCRs enriched in patients with an excellent or good prognosis were more likely to recognize cancer antigens and contained more TCR reads predicted to recognize epithelial ovarian cancer antigens. In addition, a TCR motif that is predicted to bind the TP53 neoantigen was identified, and this motif was enriched in patients with an excellent or good prognosis. Ascitic fluid in high-grade epithelial ovarian cancer patients with an excellent or good prognosis is enriched with TCRs that may recognize ovarian cancer-specific neoantigens, including mutated TP53 and TEAD1. These results suggest that an effective antigen-specific immune response in ascites is vital for a good outcome in high-grade epithelial ovarian cancer.

Microbiome Dynamics During Chemoradiation Therapy for Anal Cancer.

PURPOSE: Patients with localized squamous cell carcinoma of the anus (SCCA) who experience treatment toxicity or recurrences have few therapeutic options. Investigation into the microbiome's influence on treatment toxicity and its potential use as a predictive biomarker could improve these patients' outcomes. Our study presents the first longitudinal characterization of the SCCA tumor microbiome and its associations with treatment-related toxicities. METHODS AND MATERIALS: This prospective cohort study included patients with nonmetastatic SCCA receiving standard-of-care chemoradiation therapy. Anorectal swabs of the tumor site were collected before, during, and after treatment. Patient-reported quality-of-life metrics were collected at similar time points. 16S rRNA gene sequencing was used to perform diversity and taxonomic characterization of the SCCA microbiome. Wilcoxon signed-rank tests were used to compare microbial diversity and abundance over time. Wilcoxon rank-sum tests were used to compare microbial profiles of high and low toxicity groups. RESULTS: Twenty-two patients with SCCA were included in this study with a median age of 58.5 years (range, 39-77 years), and 18 (82%) were women. Alpha diversity remained relatively stable throughout chemoradiation therapy except for decreases in the Observed Features (P = .03) index at week 5 relative to baseline. Tumor microbial compositions changed significantly by the end of treatment (P = .03). Differential enrichment of bacteria at specific time points occurred during treatment, including the enrichment of Clostridia at follow-up (vs week 5, q = 0.019) and Corynebacterium at week 5 (vs baseline, q = 0.015; vs follow-up, q = 0.022). Patients experiencing high toxicity at week 5 had higher relative counts of Clostridia, Actinobacteria, and Clostridiales at baseline (P = .03 for all). CONCLUSIONS: The tumor microbiome changes during and after chemoradiation therapy, and patient-reported toxicity levels are associated with patients' microbial profiles. Further studies into these microbial characterizations and toxicity associations will elucidate the tumor microbiome's role in predicting treatment-related outcomes for patients with SCCA.

Expansion of Candidate HPV-Specific T Cells in the Tumor Microenvironment during Chemoradiotherapy Is Prognostic in HPV16+ Cancers.

Human papillomavirus (HPV) infection causes 600,000 new cancers worldwide each year. HPV-related cancers express the oncogenic proteins E6 and E7, which could serve as tumor-specific antigens. It is not known whether immunity to E6 and E7 evolves during chemoradiotherapy or affects survival. Using T cells from 2 HPV16+ patients, we conducted functional T-cell assays to identify candidate HPV-specific T cells and common T-cell receptor motifs, which we then analyzed across 86 patients with HPV-related cancers. The HPV-specific clones and E7-related T-cell receptor motifs expanded in the tumor microenvironment over the course of treatment, whereas non-HPV-specific T cells did not. In HPV16+ patients, improved recurrence-free survival was associated with HPV-responsive T-cell expansion during chemoradiotherapy.

Prevalence of suicidality, depression, post-traumatic stress disorder, and anxiety among female sex workers: a systematic review and meta-analysis.

The purpose was to assess prevalence of suicidality, depression, post-traumatic stress disorder (PTSD), and anxiety among female sex workers (FSW). A systematic review and meta-analysis was performed. Search strategy was performed in MEDLINE, Scopus, Web of Science, EMBASE, Ovid and Cochrane Central Database from inception until March 2020. Considered for inclusion were cross-sectional studies performed on FSW that assessed prevalence of any of the following: suicide attempt or suicidal ideation, depression, PTSD, or anxiety. Five reviewers, independently and in duplicate, selected all eligible articles in an abstract and full-text screening phase and, moreover, extracted information from each study. A binomial-normal generalized linear mixed model was employed to estimate prevalence of the conditions. From 8035 studies yielded in the search strategy, 55 were included for analysis. The overall prevalence of suicidal ideation and attempt was 27% (95% C.I. 18-39%) and 20% (95% C.I. 13-28%), respectively. Furthermore, overall prevalence of depression and PTSD was 44% (95% C.I. 35-54%) and 29% (95% C.I. 18-44%), respectively. Eleven studies were classified as high quality. Findings indicate that there is an overall high prevalence of suicidality, depression, and PTSD among FSW. Development of accessible large-scale interventions that assess mental health among this population remains critical.

Diversity and composition of gut microbiome of cervical cancer patients: Do results of 16S rRNA sequencing and whole genome sequencing approaches align?

BACKGROUND: Next generation sequencing has progressed rapidly, characterizing microbial communities beyond culture-based or biochemical techniques. 16S ribosomal RNA gene sequencing (16S) produces reliable taxonomic classifications and relative abundances, while shotgun metagenome sequencing (WMS) allows higher taxonomic and functional resolution at greater cost. The purpose of this study was to determine if 16S and WMS provide congruent information for our patient population from paired fecal microbiome samples. RESULTS: Comparative indices were highly congruent between 16S and WMS. The most abundant genera for 16S and WMS data did not overlap. Overlap was observed at the Phylum level, as expected. However, relative abundances correlated poorly between the two methodologies (all P-value>0.05). Hierarchical clustering of both sequencing analyses identified overlapping enterotypes. Both approaches were in agreement with regard to demographic variables. CONCLUSION: Diversity, evenness and richness are comparable when using 16S and WMS techniques, however relative abundances of individual genera are not. Clinical associations with diversity and evenness metrics were similarly identified with WMS or 16S.

Predictors of the length of stay of psychiatric inpatients: protocol for a systematic review and meta-analysis.

BACKGROUND: Length of stay (LOS) for inpatient psychiatric services is an important factor with serious drawbacks when it is extended more than needed. Impacts on economy, social functioning, and stigma can hamper improvement and affect the patients' experiences on future mental healthcare. Predictions of which patients have a higher chance for prolonged LOS have been extensively researched. Previous systematic reviews found consistent predictors of both longer and shorter LOS. However, they do not provide an estimate from the pooled effect sizes. Furthermore, to our knowledge, there is no meta-analysis on the influence of these factors. The primary objective of this study will be to provide point estimates on the effect sizes of all studied predictors of the LOS of psychiatric inpatients. METHODS: We will conduct a systematic search in PubMed, MEDLINE, EMBASE, and PsycINFO for observational studies evaluating the effect size of independent factors on the length of stay of psychiatric inpatients. Prospective and retrospective cohorts that assess the influence of predictors through the reporting of standardized regression coefficients will be included. We will provide a qualitative synthesis of the findings from each study and perform a meta-analysis from pooled regression coefficients that were adjusted for other variables or confounders in order to obtain a point estimate and confidence interval for all factors extracted from the included studies. DISCUSSION: The results from this study may provide more accurate predictions for mental health institutions, psychiatrists, mental health service providers, patients, and families on the prognosis regarding the length of stay for needed inpatient care. This information may be used to anticipate individuals with a higher chance for prolonged hospitalization to plan the necessary interventions for these specific situations. Considering both the benefits and disadvantages of longer and shorter stays, the pooled estimates for independent factors may be used by mental healthcare providers and patients for informed decision-making. The results from this study will also update results presented in previous studies and identify the strengths and limitations from the current available evidence. SYSTEMATIC REVIEW REGISTRATION: PROSPERO ID CRD42020172840.

A prospective study of the adaptive changes in the gut microbiome during standard-of-care chemoradiotherapy for gynecologic cancers.

BACKGROUND: A diverse and abundant gut microbiome can improve cancer patients' treatment response; however, the effect of pelvic chemoradiotherapy (CRT) on gut diversity and composition is unclear. The purpose of this prospective study was to identify changes in the diversity and composition of the gut microbiome during and after pelvic CRT. MATERIALS AND METHODS: Rectal swabs from 58 women with cervical, vaginal, or vulvar cancer from two institutions were prospectively analyzed before CRT (baseline), during CRT (weeks 1, 3, and 5), and at first follow-up (week 12) using 16Sv4 rRNA gene sequencing of the V4 hypervariable region of the bacterial 16S rRNA marker gene. 42 of these patients received antibiotics during the study period. Observed operational taxonomic units (OTUs; representative of richness) and Shannon, Simpson, Inverse Simpson, and Fisher diversity indices were used to characterize alpha (within-sample) diversity. Changes over time were assessed using a paired t-test, repeated measures ANOVA, and linear mixed modeling. Compositional changes in specific bacteria over time were evaluated using linear discriminant analysis effect size. RESULTS: Gut microbiome richness and diversity levels continually decreased throughout CRT (mean Shannon diversity index, 2.52 vs. 2.91; all P <0.01), but were at or near baseline levels in 60% of patients by week 12. Patients with higher gut diversity at baseline had the steepest decline in gut microbiome diversity. Gut microbiome composition was significantly altered during CRT, with increases in Proteobacteria and decreases in Clostridiales, but adapted after CRT, with increases in Bacteroides species. CONCLUSION: After CRT, the diversity of the gut microbiomes in this population tended to return to baseline levels by the 12 week follow-up period, but structure and composition remained significantly altered. These changes should be considered when designing studies to analyze the gut microbiome in patients who receive pelvic CRT for gynecologic cancers.

Gut microbiome diversity is an independent predictor of survival in cervical cancer patients receiving chemoradiation.

Diversity of the gut microbiome is associated with higher response rates for cancer patients receiving immunotherapy but has not been investigated in patients receiving radiation therapy. Additionally, current studies investigating the gut microbiome and outcomes in cancer patients may not have adjusted for established risk factors. Here, we sought to determine if diversity and composition of the gut microbiome was independently associated with survival in cervical cancer patients receiving chemoradiation. Our study demonstrates that the diversity of gut microbiota is associated with a favorable response to chemoradiation. Additionally, compositional variation among patients correlated with short term and long-term survival. Short term survivor fecal samples were significantly enriched in Porphyromonas, Porphyromonadaceae, and Dialister, whereas long term survivor samples were significantly enriched in Escherichia Shigella, Enterobacteriaceae, and Enterobacteriales. Moreover, analysis of immune cells from cervical tumor brush samples by flow cytometry revealed that patients with a high microbiome diversity had increased tumor infiltration of CD4+ lymphocytes as well as activated subsets of CD4 cells expressing ki67+ and CD69+ over the course of radiation therapy. Modulation of the gut microbiota before chemoradiation might provide an alternative way to enhance treatment efficacy and improve treatment outcomes in cervical cancer patients.

The Prognostic Impact of Body Composition for Locally Advanced Breast Cancer Patients Who Received Neoadjuvant Chemotherapy.

Our previous study indicated that a high amount of visceral adipose tissue was associated with poor survival outcomes in patients with early breast cancer who received neoadjuvant chemotherapy. However, inconsistency was observed in the prognostic role of body composition in breast cancer treatment outcomes. In the present study, we aimed to validate our previous research by performing a comprehensive body composition analysis in patients with a standardized clinical background. We included 198 patients with stage III breast cancer who underwent neoadjuvant chemotherapy between January 2007 and June 2015. The impact of body composition on pathologic complete response and survival outcomes was determined. Body composition measurements had no significant effect on pathologic complete response. Survival analysis showed a low ratio of total visceral adipose tissue to subcutaneous adipose tissue (V/S ratio ≤ 34) was associated with shorter overall survival. A changepoint method determined that a V/S ratio cutoff of 34 maximized the difference in overall survival. Our study indicated the prognostic effect of body composition measurements in patients with locally advanced breast cancer compared to those with early breast cancer. Further investigation will be needed to clarify the biological mechanism underlying the association of V/S ratio with prognosis in locally advanced breast cancer.

Effect of Antibiotics on Gut and Vaginal Microbiomes Associated with Cervical Cancer Development in Mice.

Antibiotics affect microbial diversity in the gut, leading to dysbiosis and impaired immunity. However, the impact of antibiotics on microbial communities at other sites, such as vagina is less understood. It is also not clear whether changes induced by antibiotics in both microbiomes affect the development of cervical cancer. In this study, we utilized the murine model to evaluate these questions. We show that oral application of broad-spectrum antibiotics in mice changed not only diversity, but composition and sharing of gut and vaginal microbiomes in mice and influenced cervical cancer development in an orthotopic tumor model. Antibiotics decreased richness and diversity indexes in the gut but increased them in the vagina. Some beneficial taxa, such as Bacteroides, Ruminococcaceae, and Lachnospiraceae increased their abundance in the vagina while other pathogenic species, such as Proteobacteria, were decreased. As a result of the changes, mice with greater richness and diversity of the vaginal microbiome after antibiotics exposure were less likely developed tumors. No association between richness and diversity of the gut microbiome and tumor development was identified.

Preliminary efficacy and tolerability profiles of first versus second-generation Long-Acting Injectable Antipsychotics in schizophrenia: A systematic review and meta-analysis.

We performed a systematic review and meta-analysis of the efficacy and safety of second generation (SG) long-acting antipsychotics (LAIAs) versus first generation (FG) LAIAs in schizophrenia. We conducted a comprehensive search in PubMed, MEDLINE, EMBASE and PsycINFO until May 2019. Inclusion criteria for randomized trials included: (1) patients ≥18 years with schizophrenia, (2) efficacy evaluated through the Positive and Negative Syndrome Scale (PANSS), (3) safety assessment through clinimetry, laboratory analysis, somatometry or adverse events, and (4) treatment duration ≥12 weeks. Data was synthesized using mean differences (MD) for continuous outcomes and risk ratios (RR) for dichotomous outcomes using a random-effect model. Of 1872 citations, 17 trials were included, and direct comparisons of SG vs FG-LAIAs were observed in 3 (n = 459). SG and FG-LAIAs had similar effects on PANSS scores (MD -1.35; 95% CI -8.33-5.64), tardive dyskinesia (RR 0.99; 95% CI, 0.47-2.07), all-cause discontinuation (RR 1.01; 95% CI 0.75-1.36), discontinuation due to inadequate efficacy (RR 1.13; 95% CI 0.81-1.59) or adverse events (RR 1.08; 95% CI 0.55-2.11). SG-LAIAs reduced the risk of using antiparkinsonian drugs (RR 0.54; 95% CI 0.54-0.76) but significantly increased serum prolactin, weight and BMI. For long-term management, depot preparations of paliperidone, haloperidol, risperidone and fluphenazine were equally effective at symptom control and adherence, with significant differences in their safety profiles. These results however are considerably limited due to the small number of included studies and are therefore preliminary, not generalizable. More clinical trials are required to obtain a broader perspective of SG-LAIAs compared to FG-LAIAs.

Gut microbial diversity and genus-level differences identified in cervical cancer patients versus healthy controls.

OBJECTIVES: The aim of this study was to characterize variation in the gut microbiome of women with locally advanced cervical cancer and compare it to healthy controls. METHODS: We characterized the 16S rDNA fecal microbiome in 42 cervical cancer patients and 46 healthy female controls. Shannon diversity index (SDI) was used to evaluate alpha (within sample) diversity. Beta (between sample) diversity was examined using principle coordinate analysis (PCoA) of unweighted Unifrac distances. Relative abundance of microbial taxa was compared between samples using Linear Discriminant Analysis Effect Size (LEfSe). RESULTS: Within cervical cancer patients, bacterial alpha diversity was positively correlated with age (p = 0.22) but exhibited an inverse relationship in control subjects (p < 0.01). Alpha diversity was significantly higher in cervical cancer patients as compared to controls (p < 0.05), though stratification by age suggested this relationship was restricted to older women (>50 years; p < 0.01). Beta diversity (unweighted Unifrac; p < 0.01) also significantly differed between cervical cancer patients and controls. Based on age- and race-adjusted LEfSe analysis, multiple taxa significantly differed between cervical cancer patients and controls. Prevotella, Porphyromonas, and Dialister were significantly enriched in cervical cancer patients, while Bacteroides, Alistipes and members of the Lachnospiracea family were significantly enriched in healthy subjects. CONCLUSION: Our study suggests differences in gut microbiota diversity and composition between cervical cancer patients and controls. Associations within the gut microbiome by age may reflect etiologic/clinical differences. These findings provide rationale for further study of the gut microbiome in cervical cancer.

Prion protein with an insertional mutation accumulates on axonal and dendritic plasmalemma and is associated with distinctive ultrastructural changes.

Prion diseases are fatal neurological diseases characterized by central nervous system deposition of abnormal forms of a membrane glycoprotein designated PrP (prion protein). Tg(PG14) transgenic mice express PrP that harbor a nine-octapeptide insertional mutation homologous to one described in a familial prion disease of humans. Tg(PG14) mice spontaneously develop a fatal neurological illness accompanied by massive apoptosis of cerebellar granule neurons and accumulation of an aggregated and weakly protease-resistant form of PrP that is not infectious. Previous light microscopic analyses of these mice left open questions regarding the subcellular distribution of the mutant protein and the nature of the neuropathological lesions produced. To address these questions, we undertook an immunogold electron microscopic study of Tg(PG14) mice. We found that mutant PrP is localized primarily on the plasma membrane of dendrites and unmyelinated axons in the hippocampus and cerebellum, with little labeling of either neuronal cell bodies or intracellular organelles. PrP deposits were shown to be associated with degenerative changes in dendritic structure. We also describe for the first time marked pathology in myelinated axons, and alterations in the axon/oligodendrocyte interface. Taken together, our results suggest cellular mechanisms by which mutant PrPs produce pathology. In addition, they highlight distinctions between familial and infectious prion disorders at the ultrastructural level that correlate with differences in cellular trafficking of the disease-associated PrP forms.

GFP-tagged mutant prion protein forms intra-axonal aggregates in transgenic mice.

A nine-octapeptide insertional mutation in the prion protein (PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular localization of this mutant PrP (designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo. Tg(PG14-EGFP) mice develop an ataxic neurological illness characterized by astrogliosis, PrP aggregation, and accumulation of a partially protease-resistant form of the mutant PrP. Strikingly, PG14-EGFP forms numerous fluorescent aggregates in the neuropil and white matter of multiple brain regions. These aggregates are particularly prominent along axonal tracts in both brain and peripheral nerve, and similar intracellular deposits are visible along the processes of cultured neurons. Our results reveal intra-axonal aggregates of a mutant PrP, which could contribute to the pathogenesis of familial prion disease by disrupting axonal transport.

Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies.

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP(C)) into an infectious, aggregated isoform (PrP(Sc)) that has a high content of beta-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP(Sc) but not PrP(C), as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19-33, 89-112, and 136-158) have been previously reported to react specifically with infectious PrP(Sc) but not PrP(C). In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.

Multiple biochemical similarities between infectious and non-infectious aggregates of a prion protein carrying an octapeptide insertion.

A nine-octapeptide insertion in the prion protein (PrP) gene is associated with an inherited form of human prion disease. Transgenic (Tg) mice that express the mouse homolog of this mutation (designated PG14) spontaneously accumulate in their brains an insoluble and weakly protease-resistant form of the mutant protein. This form (designated PG14(Spon)) is highly neurotoxic, but is not infectious in animal bioassays. In contrast, when Tg(PG14) mice are inoculated with the Rocky Mountain Laboratory (RML) strain of prions, they accumulate a different form of PG14 PrP (designated PG14(RML)) that is highly protease resistant and infectious in animal transmission experiments. We have been interested in characterizing the molecular properties of PG14(Spon) and PG14(RML), with a view to identifying features that determine two, apparently distinct properties of PrP aggregates: their infectivity and their pathogenicity. In this paper, we have subjected PG14(Spon) and PG14(RML) to a panel of assays commonly used to distinguish infectious PrP (PrP(Sc)) from cellular PrP (PrP(C)), including immobilized metal affinity chromatography, precipitation with sodium phosphotungstate, and immunoprecipitation with PrP(C)- and PrP(Sc)-specific antibodies. Surprisingly, we found that aggregates of PG14(Spon) and PG14(RML) behave identically to each other, and to authentic PrP(Sc), in each of these biochemical assays. PG14(Spon) however, in contrast to PG14(RML) and PrP(Sc), was unable to seed the misfolding of PrP(C) in an in vitro protein misfolding cyclic amplification reaction. Collectively, these results suggest that infectious and non-infectious aggregates of PrP share common structural features accounting for their toxicity, and that self-propagation of PrP involves more subtle molecular differences.