Lack of evidence that progesterone in ovulatory cycles causes breast cancer.

A recent Perspective article asserted that progesterone secretion during ovulatory cycles is the cause of breast cancer. However, we challenge most of the evidence developed in this publication. First, there is a lack of evidence that progesterone is mutagenic for breast cells. Cause of a cancer should mean initiation by mutation, as opposed to promotion. Second, subclinical ovulatory disturbances occur rather frequently in normal-length menstrual cycles. Third, the authors attribute a potential carcinogenic effect to progesterone secreted during menstrual cycles but not to progesterone during pregnancy. They did not discuss breast cancer evidence from progesterone/progestin therapeutics. They argue that in genetic primary amenorrhea, a hypothetic lower risk of breast cancer could be due to the lack of progesterone, despite the progesterone/progestin in hormone replacements these women receive. Fourth, they advocate a regulatory effect of progesterone on several genes potentially involved in cancer genesis. In particular, they attribute a lower risk of breast cancer in women with Mayer-Rokitansky-Küster-Hauser syndrome to a defect in the progesterone-stimulated Wnt4 gene. However, this defect is only present in a small subset. Thus, the postulated progesterone breast cancer risk is unconvincing, which we discuss point by point in this commentary.

Monoclonal Antibodies Opsonize Burkholderia spp. and Reduce Intracellular Actin Tail Formation in a Macrophage Infection Assay.

Melioidosis is a neglected tropical disease caused by the bacterium Burkholderia pseudomallei. The bacterium is intrinsically resistant to various antibiotics, and melioidosis is therefore difficult to treat successfully without a relapse in infection. B. pseudomallei is an intracellular pathogen and therefore, to eradicate the infection, antimicrobials must be able to access bacteria in an intracellular niche. This study assessed the ability of a panel of monoclonal antibodies (MAbs) to opsonize Burkholderia species and determine the effect that each antibody has on bacterial virulence in vitro. Murine macrophage infection assays demonstrated that monoclonal antibodies against the capsule of B. pseudomallei are opsonizing. Furthermore, one of these monoclonal antibodies reduced bacterial actin tail formation in our in vitro assays, indicating that antibodies could reduce the intracellular spread of Burkholderia thailandensis. The data presented in this paper demonstrate that monoclonal antibodies are opsonizing and can decrease bacterial actin tail formation, thus decreasing their intracellular spread. These data have informed selection of an antibody for development of an antibody-antibiotic conjugate (AAC) for melioidosis. IMPORTANCE Melioidosis is difficult to treat successfully due to the causal bacterium being resistant to many classes of antibiotics, therefore limiting available therapeutic options. New and improved therapies are urgently required to treat this disease. Here, we have investigated the potential of monoclonal antibodies to target this intracellular pathogen. We have demonstrated that monoclonal antibodies can target the bacterium, increase uptake into macrophages, and reduce actin tail formation required by the bacterium for spread between cells. Through targeting the bacterium with antibodies, we hope to disarm the pathogen, reducing the spread of infection. Ultimately, we aim to use an opsonizing antibody to deliver antibiotics intracellularly by developing an antibody-antibiotic conjugate therapeutic for melioidosis.

An Outer Membrane Vesicle-Adjuvanted Oral Vaccine Protects Against Lethal, Oral Salmonella Infection.

Non-typhoidal salmonellosis, caused by Salmonella enterica serovar Typhimurium is a common fecal-oral disease characterized by mild gastrointestinal distress resulting in diarrhea, chills, fever, abdominal cramps, head and body aches, nausea, and vomiting. Increasing incidences of antibiotic resistant invasive non-typhoidal Salmonella infections makes this a global threat requiring novel treatment strategies including next-generation vaccines. The goal of the current study was to formulate a novel vaccine platform against Salmonella infection that could be delivered orally. To accomplish this, we created a Salmonella-specific vaccine adjuvanted with Burkholderia pseudomallei outer membrane vesicles (OMVs). We show that adding OMVs to a heat-killed oral Salmonella vaccine (HKST + OMVs) protects against a lethal, oral challenge with Salmonella. Further, we show that opsonizing anti-Salmonella antibodies are induced in response to immunization and that CD4 T cells and B cells can be induced when OMVs are used as the oral adjuvant. This study represents a novel oral vaccine approach to combatting the increasing problem of invasive Salmonella infections.

Low Field Nano-NMR via Three-Level System Control.

Conventional control strategies for nitrogen-vacancy centers in quantum sensing are based on a two-level model of their triplet ground state. However, this approach fails in regimes of weak bias magnetic fields or strong microwave pulses, as we demonstrate. To overcome this limitation, we propose a novel control sequence that exploits all three levels by addressing a hidden Raman configuration with microwave pulses tuned to the zero-field transition. We report excellent performance in typical dynamical decoupling sequences, opening up the possibility for nano-NMR operation in low field environments.

Simulated effects of early menopausal bone mineral density preservation on long-term fracture risk: a feasibility study.

Prevention of early menopausal bone loss may reduce the future burden of osteoporosis. In this modelling exercise, an osteoporosis prevention strategy involving 5-year infusions of zoledronic acid, beginning early in menopause, reduced long-term fracture risk and the proportion of aging women with femoral neck densitometric osteoporosis. This strategy warrants further evaluation. INTRODUCTION: Preventing early menopausal bone loss may substantially reduce the future burden of osteoporosis. We modelled the effects of infrequent zoledronic acid infusions on long-term fracture risk. METHODS: Data from the Canadian Multicentre Osteoporosis Study (CaMos) were used to determine the expected natural history of femoral neck areal bone mineral density (BMD) and fracture risk (using FRAX®) from ages 50-80 for women with no antiresorptive drug exposures. We modelled the effects of three infusions of zoledronic acid (at ages 50, 55, 60) on long-term fracture risk, assuming this intervention would preserve BMD until age 65 years, followed by losses mirroring early menopausal BMD loss. RESULTS: At age 65, untreated women and zoledronic acid recipients had expected mean (SD) femoral neck T-scores of - 1.5(1.0) and - 0.8(1.0), 10-year major osteoporotic fracture (MOF) risks of 9.8%(5.0) and 8.0%(3.7) and hip fracture risks of 1.7%(2.4) and 0.8%(1.2), respectively. At age 80, untreated women and zoledronic acid recipients had expected femoral neck T-scores of - 1.9(0.9) and - 1.4(0.9), MOF risks of 17.9%(8.2) and 14.9%(6.4) and hip fracture risks of 6.3%(6.2) and 4.4%(4.5), respectively. The expected proportion of women with femoral neck T-score ≤ - 2.5 was 14.9% for untreated women and 3.8% for zoledronic acid recipients at age 65, increasing to 28.1% and 12.0%, respectively, at age 80. Numbers-needed-to-treat to prevent one case of densitometric osteoporosis were 9 at age 65 and 5 at age 80. CONCLUSION: Infrequent infusions of zoledronic acid, initiated early in menopause, are expected to reduce long-term fracture risk and result in a substantial reduction in the proportion of women with densitometric osteoporosis after age 65.

Bacterial-Derived Outer Membrane Vesicles are Potent Adjuvants that Drive Humoral and Cellular Immune Responses.

Discovery and development of novel adjuvants that can improve existing or next generation vaccine platforms have received considerable interest in recent years. In particular, adjuvants that can elicit both humoral and cellular immune responses would be particularly advantageous because the majority of licensed vaccines are formulated with aluminum hydroxide (alum) which predominantly promotes antibodies. We previously demonstrated that bacterial-derived outer membrane vesicles (OMV) possess inherent adjuvanticity and drive antigen-specific antibody and cellular immune responses to OMV components. Here, we investigated the ability of OMVs to stimulate innate and adaptive immunity and to function as a stand-alone adjuvant. We show that OMVs are more potent than heat-inactivated and live-attenuated bacteria in driving dendritic cell activation in vitro and in vivo. Mice immunized with OMVs admixed with heterologous peptides generated peptide-specific CD4 and CD8 T cells responses. Notably, OMV adjuvant induced much greater antibody and B cell responses to co-delivered ovalbumin compared to the responses elicited by the adjuvants alum and CpG DNA. Additionally, pre-existing antibodies raised against the OMVs did not impair OMV adjuvanticity upon repeat immunization. These results indicate that vaccines adjuvanted with OMVs elicit robust cellular and humoral immune responses, supporting further development of OMV adjuvant for use in next-generation vaccines.

Differences in fracture prevalence and in bone mineral density between Chinese and White Canadians: the Canadian Multicentre Osteoporosis Study (CaMos).

Fracture determinants differ between Canadians of Chinese and White descent, the former constituting the second largest visible minority group in Canada. The results of this study support the importance of characterizing bone health predictors in Canadians of different ethnicity to improve population-specific fracture prevention and treatment strategies. PURPOSE: We aimed to compare clinical risk factors, bone mineral density, prevalence of osteoporosis, and fractures between Chinese and White Canadians to identify ethnicity-specific risks. METHODS: We studied 236 Chinese and 8945 White Canadians aged 25+ years from the Canadian Multicentre Osteoporosis Study (CaMos). The prevalence of osteoporosis using ethnicity-specific peak bone mass (PBM), and of prior and incident low trauma fractures were assessed and compared between groups. Linear regressions, adjusting for age and anthropometric measures, were used to examine the association between baseline and 5-year changes in BMD and ethnicity. RESULTS: Chinese participants had shorter stature, lower BMI, and lower rate of falls than White participants. Adjusted models showed no significant differences in baseline BMD between ethnic groups except in younger men where total hip BMD was 0.059 g/cm2 (0.009; 0.108) lower in Chinese. Adjusted 5-year BMD change at lumbar spine was higher in older Chinese women and men compared with Whites. When using Chinese-specific PBM, the prevalence of osteoporosis in Chinese women was 2-fold lower than when using that of White women The prevalence of fractures was higher in White women compared with Chinese with differences up to 14.5% (95% CI 9.2; 19.7) and 10.5% (95% CI 4.5-16.4) in older White men. Incident fractures were rare in young Chinese compared with White participants and not different in the older groups. CONCLUSION: Our results support the importance of characterizing bone strength predictors in Chinese Canadians and the development of ethnicity-specific fracture prediction and prevention strategies.

177Lu radiolabeling and preclinical theranostic study of 1C1m-Fc: an anti-TEM-1 scFv-Fc fusion protein in soft tissue sarcoma.

PURPOSE: TEM-1 (tumor endothelial marker-1) is a single-pass transmembrane cell surface glycoprotein expressed at high levels by tumor vasculature and malignant cells. We aimed to perform a preclinical investigation of a novel anti-TEM-1 scFv-Fc fusion antibody, 1C1m-Fc, which was radiolabeled with 177Lu for use in soft tissue sarcomas models. METHODS: 1C1m-Fc was first conjugated to p-SCN-Bn-DOTA using different excess molar ratios and labeled with 177Lu. To determine radiolabeled antibody immunoreactivity, Lindmo assays were performed. The in vivo behavior of [177Lu]Lu-1C1m-Fc was characterized in mice bearing TEM-1 positive (SK-N-AS) and negative (HT-1080) tumors by biodistribution and single-photon emission SPECT/CT imaging studies. Estimated organ absorbed doses were obtained based on biodistribution results. RESULTS: The DOTA conjugation and the labeling with 177Lu were successful with a radiochemical purity of up to 95%. Immunoreactivity after radiolabeling was 86% ± 4%. Biodistribution showed a specific uptake in TEM-1 positive tumor versus liver as critical non-specific healthy organ, and this specificity is correlated to the number of chelates per antibody. A 1.9-fold higher signal at 72 h was observed in SPECT/CT imaging in TEM-1 positive tumors versus control tumors. CONCLUSION: TEM-1 is a promising target that could allow a theranostic approach to soft-tissue sarcoma, and 1C1m-Fc appears to be a suitable targeting candidate. In this study, we observed the influence of the ratio DOTA/antibody on the biodistribution. The next step will be to investigate the best conjugation to achieve an optimal tumor-to-organ radioactivity ratio and to perform therapy in murine xenograft models as a prelude to future translation in patients.

Effects of progesterone therapy on serum sclerostin levels in healthy menopausal women: a 3-month randomized, placebo-controlled clinical trial.

Sclerostin, a natural hormone made in bone, suppresses bone formation. Sclerostin is also decreased by estrogen. Progesterone, estrogen's menstrual partner, stimulates bone formation. It is unclear whether progesterone influences sclerostin. This study showed that progesterone did not change sclerostin using serum remaining from a randomized progesterone hot flush therapy trial. INTRODUCTION: Progesterone and sclerostin are both endogenous hormones acting through osteoblast-origin cells and promote or suppress bone formation, respectively. Estradiol suppresses sclerostin, but progesterone, its menstrual cycle partner hormone, has unclear sclerostin relationships. We postulated that progesterone therapy would influence serum sclerostin levels. METHODS: We obtained sclerostin levels for an ethics-approved post hoc analysis. Fasting sclerostin was measured in all remaining sera from a previous 12-week randomized controlled trial (RCT) of oral micronized progesterone (progesterone) for menopausal (> 1 year after last flow) vasomotor symptoms (VMS). Women in the RCT took 300 mg progesterone at bedtime or placebo (1:1) in a trial showing progesterone significantly decreased VMS. RESULTS: Participants were healthy menopausal, primarily Caucasian (91.2%) community-dwelling women (± SD), 55.2 ± 4.6 years old with BMI 24.9 ± 2.9 kg/m2. The baseline sclerostin level in 60 women was 28.41 ± 10.47 pmol/L. Baseline sclerostin was not correlated with the run-in VMS score (r = 0.143, P = 0.294). Paired baseline and 12-week RCT data for 52 women showed serum sclerostin levels did not change related to experimental therapy (P = 0.504). Changes in final sclerostin values adjusted for baseline were progesterone (- 1.07 ± 7.96 pmol/L) and placebo (- 2.64 ± 8.70 pmol/L). In observational data (n = 60), baseline sclerostin levels correlated with the General Framingham Cardiovascular (CVD) Risk score (r = - 0.398, P = 0.003) and self-reported health by SF-36 quality of life instrument (QoL, r = - 0.331, P = 0.016). CONCLUSION: Physiological oral micronized progesterone did not stimulate nor suppress serum sclerostin levels based on post hoc analysis of RCT data. Exploratory results, however, showed sclerostin negatively correlated with CVD risk and QoL. ClinicalTrials.gov #NCT0146469.

Testing a theoretical model of imminent fracture risk in elderly women: an observational cohort analysis of the Canadian Multicentre Osteoporosis Study.

We examined the underlying relationship between fracture risk factors and their imminent risk. Results suggested that having past year fracture, worse past year general health, worse past year physical functioning, and lower past year BMD T-score directly predicted higher imminent fracture risk. Past year falls indirectly predicted imminent risk through physical functioning and general health. INTRODUCTION: This study aimed to examine direct and indirect effects of several factors on imminent (1 year) fracture risk. METHODS: Data from women age 65 and older from population-based Canadian Multicentre Osteoporosis Study were used. Predictors were identified from study years 5 and 10, and imminent fracture data (1-year fracture) came from years 6 and 11 (year 5 predicts year 6, year 10 predicts year 11). A structural equation model (SEM) was used to test the theoretical construct. General health and physical functioning were measured as latent variables using items from the 36-Item Short Form Health Survey (SF-36) and bone mineral density (BMD) T-score was a latent variable based on observed site-specific BMD data (spine L1-L4, femoral neck, total hip). Observed variables were fractures and falls. Model fit was evaluated using root mean square error of approximation (RMSEA), Tucker Lewis index (TLI), and comparative fit index (CFI). RESULTS: The analysis included 3298 women. Model fit tests showed that the SEM fit the data well; χ2(172) = 1122.10 < .001, RMSEA = .03, TLI = .99, CFI = .99. Results suggested that having past year fracture, worse past year general health, worse past year physical functioning, and lower past year BMD T-score directly predicted higher risk of fracture in the subsequent year (p < .001). Past year falls had a statistically significant but indirect effect on imminent fracture risk through physical functioning and general health (p < .001). CONCLUSIONS: We found several direct and indirect pathways that predicted imminent fracture risk in elderly women. Future studies should extend this work by developing risk scoring methods and defining imminent risk thresholds.

Quantifying deformations and strains in human intervertebral discs using Digital Volume Correlation combined with MRI (DVC-MRI).

Physical disruptions to intervertebral discs (IVDs) can cause mechanical changes that lead to degeneration and to low back pain which affects 75% of us in our lifetimes. Quantifying the effects of these changes on internal IVD strains may lead to better preventative strategies and treatments. Digital Volume Correlation (DVC) is a non-invasive technique that divides volumetric images into subsets, and measures strains by tracking the internal patterns within them under load. Applying DVC to MRIs may allow non-invasive strain measurements. However, DVC-MRI for strain measurements in IVDs has not been used previously. The purpose of this study was to quantify the strain and deformation errors associated with DVC-MRI for measurements in human IVDs. Eight human lumbar IVDs were MRI scanned (9.4 T) for a 'zero-strain study' (multiple unloaded scans to quantify noise within the system), and a loaded study (2 mm axial compression). Three DVC methodologies: Fast-Fourier transform (FFT), direct correlation (DC), and a combination of both FFT and DC approaches were compared with subset sizes ranging from 8 to 88 voxels to establish the optimal DVC methodology and settings which were then used in the loaded study. FFT + DC was the optimal method and a subset size of 56 voxels (2520 µm) was found to be a good compromise between errors and spatial resolution. Displacement and strain errors did not exceed 28 µm and 3000 microstrain, respectively. These findings demonstrate that DVC-MRI can quantify internal strains within IVDs non-invasively and accurately. The method has unique potential for assessing IVD strains within patients.

Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages.

BACKGROUND: Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. RESULTS: We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. CONCLUSIONS: A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.

Longitudinal assessment of health-related quality of life in osteoporosis: data from the population-based Canadian Multicentre Osteoporosis Study.

Little is known about the association between health-related quality of life (HRQOL) and osteoporosis in the absence of fracture, and how HRQOL may change over time. This study provides evidence of substantially reduced HRQOL in women and men with self-reported and/or BMD-confirmed osteoporosis, even in the absence of fragility fracture. INTRODUCTION: Fragility fractures have a detrimental effect on the health-related quality of life (HRQOL) of those with osteoporosis. Less is known about the association between HRQOL and osteoporosis in the absence of fracture. METHODS: Canadian Multicentre Osteoporosis Study participants completed the SF-36, a detailed health questionnaire and measures of bone mineral density (BMD) at baseline and follow-up. We report the results of participants ≥ 50 years with 10-year follow-up. Self-reported osteoporosis at baseline and BMD-based osteoporosis at follow-up were ascertained. Multivariable linear regression models were developed for baseline SF-36 domains, component summaries, and change over time, adjusting for relevant baseline information. RESULTS: Baseline data were available for 5266 women and 2112 men. Women in the osteoporosis group had substantially lower SF-36 baseline scores, particularly in the physically oriented domains, than those without osteoporosis. A similar but attenuated pattern was evident for the men. After 10-year follow-up (2797 women and 1023 men), most domain scores dropped for women and men regardless of osteoporosis status, with the exception of mentally-oriented ones. In general, a fragility fracture was associated with lower SF-36 scores and larger declines over time. CONCLUSIONS: This study provides evidence of substantially reduced HRQOL in women and men with self-reported and/or BMD-confirmed osteoporosis, even in the absence of fragility fracture. HRQOL should be thoroughly investigated even prior to fracture, to develop appropriate interventions for all stages of the disease.

Mortality risk reduction differs according to bisphosphonate class: a 15-year observational study.

In this prospective cohort of 6120 participants aged 50+, nitrogen-bisphosphonates but not non-nitrogen bisphosphonates were associated with a significant 34% mortality risk reduction compared to non-treated propensity score matched controls. These findings open new avenues for research into mechanistic pathways. INTRODUCTION: Emerging evidence suggests that bisphosphonates (BP), first-line treatment of osteoporosis, are associated with reduced risks for all-cause mortality. This study aimed to determine the association between different BP types and mortality risk in participants with or without a fracture. METHODS: A prospective cohort study of users of different BPs matched to non-users by propensity score (age, gender, co-morbidities, fragility fracture status) and time to starting the BP medication from the population-based Canadian Multicentre Osteoporosis Study from nine Canadian centres followed from 1995 to 2013. Mortality risk for bisphosphonate users vs matched non-users was assessed using pairwise multivariable Cox proportional hazards models. RESULTS: There were 2048 women and 308 men on BP and 1970 women and 1794 men who did not receive medication for osteoporosis. The relationship between BP and mortality risk was explored in three separate 1:1 propensity score-matched cohorts of BP users and no treatment (etidronate, n = 599, alendronate, n = 498, and risedronate n = 213). Nitrogen BP (n-BP) (alendronate and risedronate) was associated with lower mortality risks [pairwise HR, 0.66 (95% CI, 0.48-0.91)] while the less potent non-n-BP, etidronate, was not [pairwise HR: 0.89 (95% CI, 0.66-1.20)]. A direct comparison between n-BP and etidronate (n = 340 pairs) also suggested a better survival for n-BP [paired HR, 0.47 (95%CI, (95% CI, 031-0.70)] for n-BP vs. etidronate]. CONCLUSION: Compared to no treatment, nitrogen but not non-nitrogen bisphosphonates appear to be associated with better survival.

PET-based prognostic survival model after radiotherapy for head and neck cancer.

PURPOSE: The aims of this multicentre retrospective study of locally advanced head and neck cancer (LAHNC) treated with definitive radiotherapy were to (1) identify positron emission tomography (PET)-18F-fluorodeoxyglucose (18F-FDG) parameters correlated with overall survival (OS) in a training cohort, (2) compute a prognostic model, and (3) externally validate this model in an independent cohort. MATERIALS AND METHODS: A total of 237 consecutive LAHNC patients divided into training (n = 127) and validation cohorts (n = 110) were retrospectively analysed. The following PET parameters were analysed: SUVMax, metabolic tumour volume (MTV), total lesion glycolysis (TLG), and SUVMean for the primary tumour and lymph nodes using a relative SUVMax threshold or an absolute SUV threshold. Cox analyses were performed on OS in the training cohort. The c-index was used to identify the highly prognostic parameters. A prognostic model was subsequently identified, and a nomogram was generated. The model was externally tested in the validation cohort. RESULTS: In univariate analysis, the significant PET parameters for the primary tumour included MTV (relative thresholds from 6 to 83% and absolute thresholds from 1.5 to 6.5) and TLG (relative thresholds from 1 to 82% and absolute thresholds from 0.5 to 4.5). For the lymph nodes, the significant parameters included MTV and TLG regardless of the threshold value. In multivariate analysis, tumour site, p16 status, MTV35% of the primary tumour, and MTV44% of the lymph nodes were independent predictors of OS. Based on these four parameters, a prognostic model was identified with a c-index of 0.72. The corresponding nomogram was generated. This prognostic model was externally validated, achieving a c-index of 0.66. CONCLUSIONS: A prognostic model of OS based on primary tumour and lymph node MTV, tumour site, and p16 status was proposed and validated. The corresponding nomogram may be used to tailor individualized treatment.