Developing, characterising, and testing a humanised 3D in vitro bone model, capable of long-term osteocyte culture

Introduction: Bone research, particularly into bone drug therapeutics, requires large-scale animal studies because complete in vitro bone models do not exist; primarily due to the complex and dynamic nature of bone tissue and the inability to culture osteocytes, (the main cell responsible for bone health maintenance). With an aging population, the number of people at risk and living with debilitating and enduring bone conditions, chiefly osteoporosis, is expected to rise. An ensuing increased pressure for effective therapies, along with the movement to replace animals in research, warrants greater effort to develop in vitro models that successfully recapitulate the cellular and extracellular complexities of bone tissue. Recent developments in 3D cell culture methods have enabled the creation of a self-structuring bone model (SSBM) that possesses physiochemical characteristics comparable to native bone, furthermore, it is the first model of its kind to house osteocytes long-term. However, this method used freshly isolated rat periosteal cells for production of each batch and the high variability in the resulting cell populations decreases reproducibility and reliability. Aim: The aim of this project was to adapt and develop this SSBM culture method to utilise a secondary human osteoblast cell line to improve the reproducibility and relevance to human disease. Resulting constructs were structurally and biologically characterised, then their feasibility as a novel bone drug screening platform was investigated. Methods: The human osteoblast cell line, hFOB 1.19, was seeded on a fibrin hydrogel, cast between two calcium phosphate anchors. Resulting SSBM constructs were cultured and analysed after 4-, 8- and 12-weeks post-cell seeding. Methods traditionally used for 2D cell culture and whole tissue analysis were adapted and developed specifically for SSBM construct analysis. These methods were then used to structurally and biologically characterise SSBM constructs, as well as measure their responsivity after exposure to bone formation modulators (BFMs): vitamin D, parathyroid hormone and Betulin. Results: The hFOB 1.19 cell line successfully produced bone model constructs composed of a collagen-containing matrix that mineralised over time. Structural analysis using alizarin red and picrosirius red staining approaches along with x-ray fluorescence revealed heterogenous distribution and maturation of this mineralising matrix; demonstrative of osteoid production, and its subsequent mineralisation to bone-like tissue. Polymerase chain reaction (qPCR) analysis showed distinct changes in gene expression over a 12-week culture period, indicative of maturing osteoblasts and osteocytogenesis. Immunofluorescent (IF) staining and ELISAs further supported these findings, confirming the presence of podoplanin (PDPN) (an early osteocytic marker), as well as prolonged cell viability and activity. qPCR, ELISAs and IF staining analysis also showed that SSBM cultures were responsive to the presence to each individual BFM. Summary: A versatile, simple, and humanised osteocyte 3D culture method, with an optimised analytical pipeline, has been established. Confirmation of their responsivity to BFMs endorses the feasibility of using the method as a high-throughput assayable platform for bone research and drug development. Preliminary steps have been taken to assess the potential of 3D bioprinting to produce such a platform and its development has the potential to make a significant impact on how bone research is approached, as well as significantly reducing and replacing the reliance and use of animals in this research field

Confirmatory structural validation and refinement of the Recurrent Urinary Tract Infection Symptom Scale

AbstractObjectivesTo confirm the structural validity of the Recurrent Urinary Tract Infection Symptom Scale (RUTISS), determining whether a bifactor model appropriately fits the questionnaire's structure and identifying areas for refinement. Used in conjunction with established clinical testing methods, this patient‐reported outcome measure addresses the urgent need to validate the patient perspective.Patients and methodsA clinically and demographically diverse sample of 389 people experiencing recurrent UTI across 37 countries (96.9% female biological sex, aged 18–87 years) completed the RUTISS online. A bifactor graded response model was fitted to the data, identifying potential items for deletion if they indicated significant differential item functioning (DIF) based on sociodemographic characteristics, contributed to local item dependence or demonstrated poor fit or discrimination capability.ResultsThe final RUTISS comprised a 3‐item symptom frequency section, a 1‐item global rating of change scale and an 11‐item general ‘rUTI symptom and pain severity’ subscale with four sub‐factor domains measuring ‘urinary symptoms’, ‘urinary presentation’, ‘UTI pain and discomfort’ and ‘bodily sensations’. The bifactor model fit indices were excellent (root mean square error of approximation [RMSEA] = 0.041, comparative fit index [CFI] = 0.995, standardised root mean square residual [SRMSR] = 0.047), and the mean‐square fit statistics indicated that all items were productive for measurement (mean square fit indices [MNSQ] = 0.64 – 1.29). Eighty‐one per cent of the common model variance was accounted for by the general factor and sub‐factors collectively, and all factor loadings were greater than 0.30 and communalities greater than 0.60. Items indicated high discrimination capability (slope parameters > 1.35).ConclusionThe 15‐item RUTISS is a patient‐generated, psychometrically robust questionnaire that dynamically assesses the patient experience of recurrent UTI symptoms and pain. This brief tool offers the unique opportunity to enhance patient‐centred care by supporting shared decision‐making and patient monitoring

Psychosocial burden and healthcare disillusionment in recurrent UTI: a large-scale international survey of patient perspectives

ObjectivesRecurrent UTI (rUTI) is a debilitating health condition that is associated with persistent mental, physical, and social burdens. People living with rUTI face inconsistencies in diagnostic testing and fragmented treatment pathways alongside their symptoms, which are likely to add considerably to their illness-related burdens. This study aimed to characterize the factors negatively impacting this population using the qualitative perspectives of people living with the condition.MethodsQualitative data were collected via free-text responses using an online survey hosted by an rUTI patient advocacy website. Female participants with self-reported rUTI (n = 1,983) described the factors that were most salient to their experience of living with the condition. Data were analyzed using a coding reliability approach to thematic analysis.ResultsTwo overarching themes were identified: (1) the patient burden of rUTI, which describes the multifaceted biopsychosocial impact of the illness, and (2) healthcare disillusionment, which describes patient dissatisfaction with healthcare received, both in terms of the treatments offered and communication with healthcare professionals. The patient burden of rUTI encompassed four subordinate themes: facing ongoing uncertainty; symptom salience; sex is not simple anymore; and perceived UTI stigma. Healthcare disillusionment included three subordinate themes: discomfort with frequent antibiotic use; fragmented treatment pathways; and devalued patient perspectives.ConclusionsThe findings demonstrated that ambiguity in the diagnosis of rUTI and inconsistencies in the subsequent treatment pathway are exacerbated by poor patient–clinician communication. The extent of the female-specific burden of rUTI symptoms confirmed the harmful effects of illness-related stigma. This novel qualitative reporting of rUTI symptom burden and life impact highlights the urgent need for increased patient-centered care for those living with rUTI. More effective rUTI management could have a major impact on treatment outcomes and patient-reported psychosocial wellbeing

Colorectal cancer linkage on chromosomes 4q21, 8q13, 12q24, and 15q22

A substantial proportion of familial colorectal cancer (CRC) is not a consequence of known susceptibility loci, such as mismatch repair (MMR) genes, supporting the existence of additional loci. To identify novel CRC loci, we conducted a genome-wide linkage scan in 356 white families with no evidence of defective MMR (i.e., no loss of tumor expression of MMR proteins, no microsatellite instability (MSI)-high tumors, or no evidence of linkage to MMR genes). Families were ascertained via the Colon Cancer Family Registry multi-site NCI-supported consortium (Colon CFR), the City of Hope Comprehensive Cancer Center, and Memorial University of Newfoundland. A total of 1,612 individuals (average 5.0 per family including 2.2 affected) were genotyped using genome-wide single nucleotide polymorphism linkage arrays; parametric and non-parametric linkage analysis used MERLIN in a priori-defined family groups. Five lod scores greater than 3.0 were observed assuming heterogeneity. The greatest were among families with mean age of diagnosis less than 50 years at 4q21.1 (dominant HLOD = 4.51, α = 0.84, 145.40 cM, rs10518142) and among all families at 12q24.32 (dominant HLOD = 3.60, α = 0.48, 285.15 cM, rs952093). Among families with four or more affected individuals and among clinic-based families, a common peak was observed at 15q22.31 (101.40 cM, rs1477798; dominant HLOD = 3.07, α = 0.29; dominant HLOD = 3.03, α = 0.32, respectively). Analysis of families with only two affected individuals yielded a peak at 8q13.2 (recessive HLOD = 3.02, α = 0.51, 132.52 cM, rs1319036). These previously unreported linkage peaks demonstrate the continued utility of family-based data in complex traits and suggest that new CRC risk alleles remain to be elucidated. © 2012 Cicek et al

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort

Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression

Colorectal and other cancer risks for carriers and noncarriers from families with a DNA mismatch repair gene mutation: A Prospective Cohort Study

To determine whether cancer risks for carriers and noncarriers from families with a mismatch repair (MMR) gene mutation are increased above the risks of the general population. We prospectively followed a cohort of 446 unaffected carriers of an MMR gene mutation (MLH1, n = 161; MSH2, n = 222; MSH6, n = 47; and PMS2, n = 16) and 1,029 their unaffected relatives who did not carry a mutation every 5 years at recruitment centers of the Colon Cancer Family Registry. For comparison of cancer risk with the general population, we estimated country-, age-, and sex-specific standardized incidence ratios (SIRs) of cancer for carriers and noncarriers. Over a median follow-up of 5 years, mutation carriers had an increased risk of colorectal cancer (CRC; SIR, 20.48; 95% CI, 11.71 to 33.27; P < .001), endometrial cancer (SIR, 30.62; 95% CI, 11.24 to 66.64; P < .001), ovarian cancer (SIR, 18.81; 95% CI, 3.88 to 54.95; P < .001), renal cancer (SIR, 11.22; 95% CI, 2.31 to 32.79; P < .001), pancreatic cancer (SIR, 10.68; 95% CI, 2.68 to 47.70; P = .001), gastric cancer (SIR, 9.78; 95% CI, 1.18 to 35.30; P = .009), urinary bladder cancer (SIR, 9.51; 95% CI, 1.15 to 34.37; P = .009), and female breast cancer (SIR, 3.95; 95% CI, 1.59 to 8.13; P = .001). We found no evidence of their noncarrier relatives having an increased risk of any cancer, including CRC (SIR, 1.02; 95% CI, 0.33 to 2.39; P = .97). We confirmed that carriers of an MMR gene mutation were at increased risk of a wide variety of cancers, including some cancers not previously recognized as being a result of MMR mutations, and found no evidence of an increased risk of cancer for their noncarrier relatives

Changes in gene expression of neo-squamous mucosa after endoscopic treatment for dysplastic Barrett’s esophagus and intramucosal adenocarcinoma

Author version made available in accordance with publisher copyright policy.Abstract Background: Endoscopic therapy, including by radiofrequency ablation (RFA) or endoscopic mucosal resection (EMR), is first line treatment for Barrett’s esophagus (BE) with high-grade dysplasia (HGD) or intramucosal cancer (IMC) and may be appropriate for some patients with low-grade dysplasia (LGD). Objective: The purpose of this study was to investigate the molecular effects of endotherapy. Methods: mRNA expression of 16 genes significantly associated with different BE stages was measured in paired pretreatment BE tissues and post-treatment neo-squamous biopsies from 36 patients treated by RFA (19 patients, 3 IMC, 4 HGD, 12 LGD) or EMR (17 patients, 4 IMC, 13 HGD). EMR was performed prior to RFA in eight patients. Normal squamous esophageal tissues were from 20 control individuals. Results: Endoscopic therapy resulted in significant change towards the normal squamous expression profile for all genes. The neo-squamous expression profile was significantly different to the normal control profile for 11 of 16 genes. Conclusion: Endotherapy results in marked changes in mRNA expression, with replacement of the disordered BE dysplasia or IMC profile with a more ‘‘normal’’ profile. The neo-squamous mucosa was significantly different to the normal control squamous mucosa for most genes. The significance of this finding is uncertain but it may support continued endoscopic surveillance after successful endotherapy

Homologous recombination DNA repair defects in PALB2- associated breast cancers

Abstract: Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD

Will the Public's Health Fall Victim to the Home Foreclosure Epidemic?

Gary Bennett and colleagues discuss the ways in which the dramatic rise in home foreclosures, particularly in the US, may have health consequences

Risk of extracolonic cancers for people with biallelic and monoallelic mutations in MUTYH: Extracolonic cancer risks for people with biallelic and monoallelicMUTYHmutations

Germline mutations in the DNA base excision repair gene MUTYH are known to increase a carrier’s risk of colorectal cancer. However, the risks of other (extracolonic) cancers for MUTYH mutation carriers are not well defined. We identified 266 probands (91% Caucasians) with a MUTYH mutation (41 biallelic and 225 monoallelic) from the Colon Cancer Family Registry. Mutation status, sex, age, and histories of cancer from their 1,903 first- and 3,255 second-degree relatives, were analysed using modified segregation analysis conditioned on the ascertainment criteria. Compared with incidences for the general population, hazard ratios (HRs) (95% confidence intervals [CIs]) for biallelic MUTYH mutation carriers were: urinary bladder cancer, 19(3.7–97); and ovarian cancer, 17(2.4–115). The HRs (95%CI) for monoallelic MUTYH mutation carriers were: gastric cancer, 9.3(6.7–13); hepatobiliary cancer, 4.5(2.7–7.5); endometrial cancer, 2.1(1.1–3.9); and breast cancer, 1.4(1.0–2.0). There was no evidence for an increased risk of cancers at the other sites examined (brain, pancreas, kidney or prostate). Based on the USA population incidences, the estimated cumulative risks (95%CI) to age 70 years for biallelic mutation carriers were: bladder cancer, 25%(5%–77%) for males and 8%(2%–33%) for females; and ovarian cancer, 14%(2%–65%). The cumulative risks (95%CI) for monoallelic mutation carriers were: gastric cancer, 5%(4%–7%) for males and 2.3%(1.7%–3.3%) for females; hepatobiliary cancer, 3%(2%–5%) for males and 1.4%(0.8%–2.3%) for females; endometrial cancer, 3%(2%–6%); and breast cancer 11%(8%–16%). These unbiased estimates of both relative and absolute risks of extracolonic cancers for people, mostly Caucasians, with MUTYH mutations will be important for their clinical management