Driving delivery and uptake of catch-up vaccination among adolescent and adult migrants in UK general practice: a mixed methods pilot study.

BACKGROUND: Migrants in the UK and Europe face vulnerability to vaccine-preventable diseases (VPDs) due to missed childhood vaccines and doses and marginalisation from health systems. Ensuring migrants receive catch-up vaccinations, including MMR, Td/IPV, MenACWY, and HPV, is essential to align them with UK and European vaccination schedules and ultimately reduce morbidity and mortality. However, recent evidence highlights poor awareness and implementation of catch-up vaccination guidelines by UK primary care staff, requiring novel approaches to strengthen the primary care pathway. METHODS: The 'Vacc on Track' study (May 2021-September 2022) aimed to measure under-vaccination rates among migrants in UK primary care and establish new referral pathways for catch-up vaccination. Participants included migrants aged 16 or older, born outside of Western Europe, North America, Australia, or New Zealand, in two London boroughs. Quantitative data on vaccination history, referral, uptake, and sociodemographic factors were collected, with practice nurses prompted to deliver catch-up vaccinations following UK guidelines. Focus group discussions and in-depth interviews with staff and migrants explored views on delivering catch-up vaccination, including barriers, facilitators, and opportunities. Data were analysed using STATA12 and NVivo 12. RESULTS: Results from 57 migrants presenting to study sites from 18 countries (mean age 41 [SD 7.2] years; 62% female; mean 11.3 [SD 9.1] years in UK) over a minimum of 6 months of follow-up revealed significant catch-up vaccination needs, particularly for MMR (49 [86%] required catch-up vaccination) and Td/IPV (50 [88%]). Fifty-three (93%) participants were referred for any catch-up vaccination, but completion of courses was low (6 [12%] for Td/IPV and 33 [64%] for MMR), suggesting individual and systemic barriers. Qualitative in-depth interviews (n = 39) with adult migrants highlighted the lack of systems currently in place in the UK to offer catch-up vaccination to migrants on arrival and the need for health-care provider skills and knowledge of catch-up vaccination to be improved. Focus group discussions and interviews with practice staff (n = 32) identified limited appointment/follow-up time, staff knowledge gaps, inadequate engagement routes, and low incentivisation as challenges that will need to be addressed. However, they underscored the potential of staff champions, trust-building mechanisms, and community-based approaches to strengthen catch-up vaccination uptake among migrants. CONCLUSIONS: Given the significant catch-up vaccination needs of migrants in our sample, and the current barriers to driving uptake identified, our findings suggest it will be important to explore this public health issue further, potentially through a larger study or trial. Strengthening existing pathways, staff capacity and knowledge in primary care, alongside implementing new strategies centred on cultural competence and building trust with migrant communities will be important focus areas.

Understanding the views of adult migrants around catch-up vaccination for missed routine immunisations to define strategies to improve coverage: A UK in-depth interview study.

BACKGROUND: The World Health Organization's (WHO) Immunization Agenda 2030 emphasises ensuring equitable access to vaccination across the life course. This includes placing an emphasis on migrant populations who may have missed key childhood vaccines, doses, and boosters due to disrupted healthcare systems and the migration process, or differing vaccination schedules in home countries. Guidelines exist in the UK for offering catch-up vaccinations to adolscent and adult migrants with incomplete or uncertain vaccination status (including MMR, Td-IPV, MenACWY, HPV), but emerging evidence suggests awareness and implementation in primary care is poor. It is unclear whether patient-level barriers to uptake of catch-up vaccinations also exist. We explored experiences and views around catch-up vaccination among adult migrants from a range of backgrounds, to define strategies for improving catch-up vaccination policy and practice. METHODS: In-depth semi-structured interviews were carried out in two phases with adult migrant populations (refugees, asylum seekers, undocumented migrants, those with no recourse to public funds) on views and experiences around vaccination, involving a team of peer researchers from specific migrant communities trained through the study. In Phase 1, we conducted remote interviews with migrants resident in the UK for < 10 years, from diverse backgrounds. In Phase 2, we engaged specifically Congolese and Angolan migrants as part of a community-based participatory study. Topic guides were developed iteratively and piloted. Participants were recruited using purposive, opportunistic and snowball sampling methods. Interviews were conducted in English (interpreters offered), Lingala or French and were audio-recorded, transcribed and analysed using a thematic framework approach in NVivo 12. RESULTS: 71 participants (39 in Phase 1, 32 in Phase 2) were interviewed (Mean age 43.6 [SD:12.4] years, 69% female, mean 9.5 [SD:7] years in the UK). Aside from COVID-19 vaccines, most participants reported never having been offered vaccinations or asked about their vaccination history since arriving in the UK as adults. Few participants mentioned being offered specific catch-up vaccines (e.g. MMR/Td-IPV) when attending a healthcare facility on arrival in the UK. Vaccines such as flu vaccines, pregnancy-related or pre-travel vaccination were more commonly mentioned. In general, participants were not aware of adult catch-up vaccination but regarded it positively when it was explained. A few participants expressed concerns about side-effects, risks/inconveniences associated with access (e.g. links to immigration authorities, travel costs), preference for natural remedies, and hesitancy to engage in further vaccination campaigns due to the intensity of COVID-19 vaccination campaigns. Trust was a major factor in vaccination decisions, with distinctions noted within and between groups; some held a healthcare professional's recommendation in high regard, while others were less trusting towards the healthcare system because of negative experiences of the NHS and past experiences of discrimination, injustice and marginalisation by wider authorities. CONCLUSIONS: The major barrier to adult catch-up vaccination for missed routine immunisations and doses in migrant communities in the UK is the limited opportunities, recommendations or tailored vaccination information presented to migrants by health services. This could be improved with financial incentives for provision of catch-up vaccination in UK primary care, alongside training of healthcare professionals to support catch-up immunisation and raise awareness of existing guidelines. It will also be essential to address root causes of mistrust around vaccination, where it exists among migrants, by working closely with communities to understand their needs and meaningfully involving migrant populations in co-producing tailored information campaigns and culturally relevant interventions to improve coverage.

Defining drivers of under-immunization and vaccine hesitancy in refugee and migrant populations.

BACKGROUND/OBJECTIVE: Some refugee and migrant populations globally showed lower uptake of COVID-19 vaccines and are also considered to be an under-immunized group for routine vaccinations. These communities may experience a range of barriers to vaccination systems, yet there is a need to better explore drivers of under-immunization and vaccine hesitancy in these mobile groups. METHODS: We did a global rapid review to explore drivers of under-immunization and vaccine hesitancy to define strategies to strengthen both COVID-19 and routine vaccination uptake, searching MEDLINE, Embase, Global Health PsycINFO and grey literature. Qualitative data were analysed thematically to identify drivers of under-immunization and vaccine hesitancy, and then categorized using the 'Increasing Vaccination Model'. RESULTS: Sixty-three papers were included, reporting data on diverse population groups, including refugees, asylum seekers, labour migrants and undocumented migrants in 22 countries. Drivers of under-immunization and vaccine hesitancy pertaining to a wide range of vaccines were covered, including COVID-19 (n = 27), human papillomavirus (13), measles or Measles-mumps-rubella (MMR) (3), influenza (3), tetanus (1) and vaccination in general. We found a range of factors driving under-immunization and hesitancy in refugee and migrant groups, including unique awareness and access factors that need to be better considered in policy and service delivery. Acceptability of vaccination was often deeply rooted in social and historical context and influenced by personal risk perception. CONCLUSIONS: These findings hold direct relevance to current efforts to ensure high levels of global coverage for a range of vaccines and to ensure that marginalized refugee and migrant populations are included in the national vaccination plans of low-, middle- and high-income countries. We found a stark lack of research from low- and middle-income and humanitarian contexts on vaccination in mobile groups. This needs to be urgently rectified if we are to design and deliver effective programmes that ensure high coverage for COVID-19 and routine vaccinations.

Defining the determinants of vaccine uptake and undervaccination in migrant populations in Europe to improve routine and COVID-19 vaccine uptake: a systematic review.

Understanding why some migrants in Europe are at risk of underimmunisation and show lower vaccination uptake for routine and COVID-19 vaccines is critical if we are to address vaccination inequities and meet the goals of WHO's new Immunisation Agenda 2030. We did a systematic review (PROSPERO: CRD42020219214) exploring barriers and facilitators of vaccine uptake (categorised using the 5As taxonomy: access, awareness, affordability, acceptance, activation) and sociodemographic determinants of undervaccination among migrants in the EU and European Economic Area, the UK, and Switzerland. We searched MEDLINE, CINAHL, and PsycINFO from 2000 to 2021 for primary research, with no restrictions on language. 5259 data sources were screened, with 67 studies included from 16 countries, representing 366 529 migrants. We identified multiple access barriers-including language, literacy, and communication barriers, practical and legal barriers to accessing and delivering vaccination services, and service barriers such as lack of specific guidelines and knowledge of health-care professionals-for key vaccines including measles-mumps-rubella, diphtheria-pertussis-tetanus, human papillomavirus, influenza, polio, and COVID-19 vaccines. Acceptance barriers were mostly reported in eastern European and Muslim migrants for human papillomavirus, measles, and influenza vaccines. We identified 23 significant determinants of undervaccination in migrants (p<0·05), including African origin, recent migration, and being a refugee or asylum seeker. We did not identify a strong overall association with gender or age. Tailored vaccination messaging, community outreach, and behavioural nudges facilitated uptake. Migrants' barriers to accessing health care are already well documented, and this Review confirms their role in limiting vaccine uptake. These findings hold immediate relevance to strengthening vaccination programmes in high-income countries, including for COVID-19, and suggest that tailored, culturally sensitive, and evidence-informed strategies, unambiguous public health messaging, and health system strengthening are needed to address access and acceptance barriers to vaccination in migrants and create opportunities and pathways for offering catch-up vaccinations to migrants.

Isolation and Characterization of a ssDNA Aptamer against Major Soluble Antigen of Renibacterium salmoninarum.

Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by Renibacterium salmoninarum (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a Kd of 3.0 ± 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses.

Evidence for the involvement of interleukin-1α during development of experimental cytomegalovirus retinitis in immunosuppressed mice.

Interleukin-1α (IL-1α) is an alarmin involved in the recruitment of macrophages and neutrophils during tissue inflammation. IL-1α can undergo cleavage by proteases, such as calpain-1, that enhances IL-1α binding to its receptor, although proteolytic cleavage is not necessary for biological activity. Macrophages and neutrophils are involved in the retinal inflammation associated with development of AIDS-related human cytomegalovirus (HCMV) retinitis. We therefore performed studies to test the hypothesis that IL-1α gene expression is stimulated intraocularly during retinitis development using two mouse models of murine cytomegalovirus (MCMV) retinitis that differ in method of immunosuppression, one by retrovirus-induced immunosuppression (MAIDS) and the other by corticosteroid-induced immunosuppression. MCMV-infected eyes of groups of retinitis-susceptible mice with MAIDS of 10 weeks duration (MAIDS-10 mice) and retinitis-susceptible corticosteroid-treated mice showed significant stimulation of IL-1α mRNA. Western blot analysis confirmed IL-1α protein production within the MCMV-infected eyes of MAIDS-10 mice. Whereas significant intraocular calpain-1 mRNA and protein production were also observed within MCMV-infected eyes of MAIDS-10 mice, the MCMV-infected eyes of retinitis-susceptible corticosteroid-treated mice showed a pattern of mRNA synthesis equivalent to that found within the MCMV-infected eyes of healthy mice that fail to develop retinitis. Our findings suggest a role for the alarmin IL-1α in the pathogenesis of MCMV retinitis in immunosuppressed mice. These findings may extend to the pathogenesis of HCMV retinitis in patients with AIDS or other forms of immunosuppression.

Impact of a Smartphone App Reminder on Adherence to Aspirin Prescribed as Antithrombotic Therapy.

BACKGROUND: Many patients are discharged with aspirin (ASA) as an anticoagulant after joint replacement surgery. In studies in which ASA was prescribed, doses were frequently missed. Adherence to postoperative ASA regimen is critical to preventing thrombotic complications. This randomized controlled study evaluated the impact of an existing medication adherence smartphone application (app) on adherence to twice daily ASA 81 mg for 35 days after knee or hip arthroplasty. METHODOLOGY: Patients were randomized to either the app (intervention) group or the usual care (control) group. All patients received a baseline interview with a survey and demographics collected prior to randomization, a 36-day follow-up call for phone pill count, and a 6-week in-office interview with surveys and an in-person pill count. A convenience sample of 195 patients enrolled; 122 completed pill counts at both baseline and end of study. Ages ranged from 29 to 89 (mean: 60.4, SD: 10.1) years. The majority had a bachelor's degree or higher (59.3%), made more than $75,000 (or were retired; 51.9%), were of White race (75.9%), and female (53.8%). There were no significant demographic differences between the groups. RESULTS: There were no significant group differences in final pill counts, adherence (reasons for missed pills), or ASA Medication self-efficacy scores. However, the intervention group scored significantly higher on the ASA Adherence measure (general ease in and ability to take ASA; p = .020). Higher ASA Adherence scores were associated with lower pill counts at the end of study (better adherence) in the intervention group. There was a high rate of attrition related to failure to bring the ASA to hospital or to the follow-up appointment. CONCLUSIONS: Although there were no significant differences in final pill counts between groups, the app group reported more ease in taking pills. With smartphone use virtually ubiquitous, this project provided an opportunity to educate patients and nurses about how technology can support medication adherence. It was also an excellent opportunity to involve clinical nurses in a funded research project.

Parthanatos-associated proteins are stimulated intraocularly during development of experimental murine cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression.

The mechanisms that contribute to retinal tissue destruction during the onset and progression of AIDS-related human cytomegalovirus (HCMV) retinitis remain unclear. Evidence for the stimulation of multiple cell death pathways including apoptosis, necroptosis, and pyroptosis during the pathogenesis of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS) has been reported. Parthanatos is a caspase-independent cell death pathway mediated by rapid overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) and distinct from other cell death pathways. Using the MAIDS model of MCMV retinitis, studies were performed to test the hypothesis that intraocular MCMV infection of mice with MAIDS stimulates parthanatos-associated messenger RNAs (mRNAs) and proteins within the eye during the development of retinal necrosis that takes place by 10 days after MCMV infection. MCMV-infected eyes of MAIDS mice exhibited significant stimulation of PARP-1 mRNA and proteins at 3 days after infection but declined thereafter at 6 and 10 days after infection. Additional studies showed the intraocular stimulation of mRNAs or proteins before MCMV retinitis development for two additional participants in parthanatos, polymer of ADP-ribose and poly (ADP-ribose) glycohydrolase. These results provide new evidence for a role for parthanatos during MAIDS-related MCMV retinitis that may also extend to AIDS-related HCMV retinitis.

Randomized Controlled Trial of a Natural Food-Based Fiber Solution to Prevent Constipation in Postoperative Spine Fusion Patients.

BACKGROUND: Constipation after orthopaedic surgery occurs frequently, likely due to a combination of high levels of opioid medications for severe pain management and mobility limitations after surgery. It can result in serious complications, increased cost, and patient discomfort. PURPOSE: This study evaluated a natural food-based fiber solution to prevent constipation in postoperative orthopaedic patients. METHODS: A posttest control group-randomized study design was used. Dependent variables were presence of postoperative constipation, time to first bowel movement (BM), and total number of postoperative BMs. Descriptive statistics, Student's t tests, and Mann-Whitney nonparametric 2-group tests with chi-square analysis were used. Level of significance for all tests was p < .05. Forty-six participants were evaluated. RESULTS: Ages were similar for both the intervention and control groups. Bowel Function Index (BFI) scores were not significantly different (p = .448). No significant group differences were present for the individual BFI item scores (p > .05). The number of patients with a BM during the first 3 days was not significantly different (p = .489). There were no significant differences found between the 2 groups regarding laxative administration (p > .05 for all laxatives). CONCLUSION: Further studies are indicated that address natural fibers and pharmaceutical methods for the prevention of constipation after spinal surgery.

Impact of a Preset Daily Cell Phone Alarm on Medication Adherence for Aspirin as Antithrombotic Therapy.

The ability of patients to adhere to medication regimens is considered critical to achieving optimal results. Many patients are discharged with aspirin (ASA) as an anticoagulant for venous thrombosis embolism prophylaxis after joint replacement surgery. In studies where ASA was prescribed as an antithrombotic after selected orthopaedic surgeries, both lack of understanding and missing doses were identified as factors that affected adherence rates (D. , ). The purpose of this study was to explore the preliminary impact of a preset telephone alarm on medication adherence in adults prescribed ASA for 35 days after knee or hip arthroplasty. This was a randomized controlled trial (n = 79). Adherence was measured with a four-question self-reporting tool. Average age was 61 years. The majority were female (59.5%) and Caucasian (62.0%) with college or graduate degree-level education (78.5%). When comparing the groups, there were no significant differences between the two groups for the demographics of age, gender, and race. The alarm group had significantly better adherence rates, with fewer people who forgot to take their ASA; only 29.7% of the alarm group ever forgot to take their medication compared with 59.5% of the no-alarm group (p = .008). It seems that simple cell phone alarms can serve as effective reminders to patients to take selected medications as prescribed.

Self-Reported Rates of Adherence to Aspirin Prescribed as an Antithrombotic Therapy Following Postoperative Total Joint Replacement.

BACKGROUND: Patients may not understand the importance of medication adherence when discharged with aspirin (ASA) as an anticoagulant for venous thromboembolism (VTE) prophylaxis after joint replacement surgery. PURPOSE: The purpose of this study was to examine self-reported rates of adherence to postoperative use of ASA for VTE prophylaxis. METHODS: This was an exploratory, descriptive study using a convenience sample of 99 participants. Adherence was measured with a 4-question self-reporting tool. RESULTS: Thirty-seven percent were male and 76% were Caucasian, and median age was 63 years. Ninety-two percent had the dosage correct, 76% knew how long to take the medication, and 100% of the sample was still taking the ASA; 40% had forgotten to take 1 or more doses of the medication. No associations were noted. Overall compliance was 44%. CONCLUSION: A significant number missed 1 or more doses. This is an opportunity for nurses to work with patients on innovative ways to improve adherence.

Pro-inflammatory T-lymphocytes rapidly infiltrate into the brain and contribute to neuronal injury following cardiac arrest and cardiopulmonary resuscitation.

Although inflammatory mechanisms have been linked to neuronal injury following global cerebral ischemia, the presence of infiltrating peripheral immune cells remains understudied. We performed flow cytometry of single cell suspensions obtained from the brains of mice at varying time points after global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR) to characterize the influx of lymphocytes into the injured brain. We observed that CA/CPR caused a large influx of lymphocytes within 3h of resuscitation that was maintained for the 3day duration of our experiments. Using cell staining flow cytometry we observed that the large majority of infiltrating lymphocytes were CD4(+) T cells. Intracellular stains revealed a large proportion of pro-inflammatory T cells expressing either TNFα or INFγ. Importantly, the lack of functional T cells in TCRα knockout mice reduced neuronal injury following CA/CPR, implicating pro-inflammatory T cells in the progression of ischemic neuronal injury. Finally, we made the remarkable observation that the novel CD4(+)CD40(+) (Th40) population of pro-inflammatory T cells that are strongly associated with autoimmunity are present in large numbers in the injured brain. These data indicate that studies investigating the neuro-immune response after global cerebral ischemia should consider the role of infiltrating T cells in orchestrating the acute and sustained immune response.

A CD40-targeted peptide controls and reverses type 1 diabetes in NOD mice.

AIMS/HYPOTHESIS: The CD40-CD154 interaction directs autoimmune inflammation. Therefore, a long-standing goal in the treatment of autoimmune disease has been to control the formation of that interaction and thereby prevent destructive inflammation. Antibodies blocking CD154 are successful in mouse models of autoimmune disease but, while promising when used in humans, unfortunate thrombotic events have occurred, forcing the termination of those studies. METHODS: To address the clinical problem of thrombotic events caused by anti-CD154 antibody treatment, we created a series of small peptides based on the CD154 domain that interacts with CD40 and tested the ability of these peptides to target CD40 and prevent type 1 diabetes in NOD mice. RESULTS: We identified a lead candidate, the 15-mer KGYY15 peptide, which specifically targets CD40-positive cells in a size- and sequence-dependent manner. It is highly efficient in preventing hyperglycaemia in NOD mice that spontaneously develop type 1 diabetes. Importantly, KGYY15 can also reverse new-onset hyperglycaemia. KGYY15 is well tolerated and functions to control the cytokine profile of culprit Th40 effector T cells. The KGYY15 peptide is 87% homologous to the human sequence, suggesting that it is an important candidate for translational studies. CONCLUSIONS/INTERPRETATION: Peptide KGYY15 constitutes a viable therapeutic option to antibody therapy in targeting the CD40-CD154 interaction in type 1 diabetes. Given the involvement of CD40 in autoimmunity in general, it will also be important to evaluate KGYY15 in the treatment of other autoimmune diseases. This alternative therapeutic approach opens new avenues of exploration in targeting receptor-ligand interactions.

Defining a new biomarker for the autoimmune component of Multiple Sclerosis: Th40 cells.

Multiple Sclerosis (MS) is a chronic inflammatory, neurodegenerative disease. Diagnosis is very difficult requiring defined symptoms and multiple CNS imaging. A complicating issue is that almost all symptoms are not disease specific for MS. Autoimmunity is evident, yet the only immune-related diagnostic tool is cerebral-spinal fluid examination for oligoclonal bands. This study addresses the impact of Th40 cells, a pathogenic effector subset of helper T cells, in MS. MS patients including relapsing/remitting MS, secondary progressive MS and primary progressive MS were examined for Th40 cell levels in peripheral blood and, similar to our findings in autoimmune type 1 diabetes, the levels were significantly (p<0.0001) elevated compared to controls including healthy non-autoimmune subjects and another non-autoimmune chronic disease. Classically identified Tregs were at levels equivalent to non-autoimmune controls but the Th40/Treg ratio still predicted autoimmunity. The cohort displayed a wide range of HLA haplotypes including the GWAS identified predictive HLA-DRB1*1501 (DR2). However half the subjects did not carry DR2 and regardless of HLA haplotype, Th40 cells were expanded during disease. In RRMS Th40 cells demonstrated a limited TCR clonality. Mechanistically, Th40 cells demonstrated a wide array of response to CNS associated self-antigens that was dependent upon HLA haplotype. Th40 cells were predominantly memory phenotype producing IL-17 and IFNγ with a significant portion producing both inflammatory cytokines simultaneously suggesting an intermediary between Th1 and Th17 phenotypes.

Functional characterisation of human cells harbouring a novel t(2p;7p) translocation involving TNS3 and EXOC6B genes.

BACKGROUND: Tensin3 is an intracellular cytoskeleton-regulating protein, the loss of which is associated with increased cell motility, as has been observed in some human cancers. A novel chromosomal translocation, t(2;7)(p13;p12), present in a patient with a complex syndromic phenotype, directly involves Tensin3 (TNS3) and EXOC6B genes. This translocation could impair the expression of Tensin3 and ExoC6B proteins, and potentially produce two novel fusion transcripts. In the present study, we have investigated the expression and phenotypic features of these potential products in cultured cells from the proband. METHODS: Skin fibroblasts isolated from the proband as well as an age-matched control were grown in cell culture. Cells were used for quantitative RT-PCR, western blot and immunofluorescent confocal microscopy, which determined Tensin3 gene and protein expression. Phase-contrast and confocal microscopy additionally revealed cellular phenotype differences. A scratch wound assay monitored by live cell imaging measured cellular migration rates. RESULTS: The levels of Tensin3 at both mRNA and protein levels were lower in proband cells versus control fibroblasts. Proband cells displayed broader and shorter morphologies versus control fibroblasts, and immunofluorescent staining revealed additional Tensin3 expression along cytoskeletal filaments and the cell periphery only in control fibroblasts. In addition, proband fibroblasts showed a significantly higher migration rate than control cells over 24 h. CONCLUSIONS: The phenotypic changes observed in proband cells may arise from TNS3 haploinsufficiency, causing partial loss of full-length Tensin3 protein. These results further expose a role for Tensin3 in cytoskeletal organisation and cell motility and may also help to explain the syndromic features observed in the patient.

CpG dinucleotide-specific hypermethylation of the TNS3 gene promoter in human renal cell carcinoma.

Tensin3 is a cytoskeletal regulatory protein that inhibits cell motility. Downregulation of the gene encoding Tensin3 (TNS3) in human renal cell carcinoma (RCC) may contribute to cancer cell metastatic behavior. We speculated that epigenetic mechanisms, e.g., gene promoter hypermethylation, might account for TNS3 downregulation. In this study, we identified and validated a TNS3 gene promoter containing a CpG island, and quantified the methylation level within this region in RCC. Using a luciferase reporter assay we demonstrated a functional minimal promoter activity for a 500-bp sequence within the TNS3 CpG island. Pyrosequencing enabled quantitative determination of DNA methylation of each CpG dinucleotide (a total of 43) in the TNS3 gene promoter. Across the entire analyzed CpG stretch, RCC DNA showed a higher methylation level than both non-tumor kidney DNA and normal control DNA. Out of all the CpGs analyzed, two CpG dinucleotides, specifically position 2 and 8, showed the most pronounced increases in methylation levels in tumor samples. Furthermore, CpG-specific higher methylation levels were correlated with lower TNS3 gene expression levels in RCC samples. In addition, pharmacological demethylation treatment of cultured kidney cells caused a 3-fold upregulation of Tensin3 expression. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter occurring in human RCC, suggesting an epigenetic mechanism for aberrant Tensin downregulation in human kidney cancer.

An alternative role for Foxp3 as an effector T cell regulator controlled through CD40.

The BDC2.5 T cell clone is highly diabetogenic, but the transgenic mouse generated from that clone is surprisingly slow in diabetes development. Although defining pathogenic effector T cells in autoimmunity has been inconsistent, CD4(+) cells expressing the CD40 receptor (Th40 cells) are highly diabetogenic in NOD mice, and NOD.BDC2.5.TCR.Tg mice possess large numbers of these cells. Given the importance of CD40 for pathogenic T cell development, BDC2.5.CD40(-/-) mice were created. Regulatory T cells, CD4(+)CD25(hi)Foxp3(+), develop normally, but pathogenic effector cells are severely reduced in number. Th40 cells from diabetic BDC2.5 mice rapidly induce diabetes in NOD.scid recipients, but Th40 cells from prediabetic mice transfer diabetes very slowly. Demonstrating an important paradigm shift, effector Th40 cells from prediabetic mice are Foxp3(+). As mice age, moving to type 1 diabetes development, Th40 cells lose Foxp3. When Th40 cells that are Foxp3(+) are transferred to NOD.scid recipients, disease is delayed. Th40 cells that are Foxp3(-) rapidly transfer disease. Th40 cells from BDC2.5.CD40(-/-) mice do not transfer disease nor do they lose Foxp3 expression. Mechanistically, Foxp3(+) cells produce IL-17 but do not produce IFN-γ, whereas Foxp3(-) Th40 cells produce IFN-γ and IL-2. This poses a new consideration for the function of Foxp3, as directly impacting effector T cell function.

Oncogene-dependent control of miRNA biogenesis and metastatic progression in a model of undifferentiated pleomorphic sarcoma.

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue malignancies. Patients with large, high-grade sarcomas often develop fatal lung metastases. Understanding the mechanisms underlying sarcoma metastasis is needed to improve treatment of these patients. Micro-RNAs (miRNAs) are a class of small RNAs that post-transcriptionally regulate gene expression. Global alterations in miRNAs are frequently observed in a number of disease states including cancer. The signalling pathways that regulate miRNA biogenesis are beginning to emerge. To test the relevance of specific oncogenic mutations in miRNA biogenesis in sarcoma, we used primary soft tissue sarcomas expressing either Braf(V600E) or Kras(G12D). We found that Braf(V600E) mutant tumours, which have increased MAPK signalling, have higher levels of mature miRNAs and enhanced miRNA processing. To investigate the relevance of oncogene-dependent alterations in miRNA biogenesis, we introduced conditional mutations in Dicer and showed that Dicer haploinsufficiency promotes the development of distant metastases in an oncogene-dependent manner. These results demonstrate that a specific oncogenic mutation can cooperate with mutation in Dicer to promote tumour progression in vivo.

A novel imaging system permits real-time in vivo tumor bed assessment after resection of naturally occurring sarcomas in dogs.

BACKGROUND: Treatment of soft tissue sarcoma (STS) includes complete tumor excision. However, in some patients, residual sarcoma cells remain in the tumor bed. We previously described a novel hand-held imaging device prototype that uses molecular imaging to detect microscopic residual cancer in mice during surgery. QUESTIONS/PURPOSES: To test this device in a clinical trial of dogs with naturally occurring sarcomas, we asked: (1) Are any adverse clinical or laboratory effects observed after intravenous administration of the fluorescent probes? (2) Do canine sarcomas exhibit fluorescence after administration of the cathepsin-activated probe? (3) Is the tumor-to-background ratio sufficient to distinguish tumor from tumor bed? And (4) can residual fluorescence be detected in the tumor bed during surgery and does this correlate with a positive margin? METHODS: We studied nine dogs undergoing treatment for 10 STS or mast cell tumors. Dogs received an intravenous injection of VM249, a fluorescent probe that becomes optically active in the presence of cathepsin proteases. After injection, tumors were removed by wide resection. The tumor bed was imaged using the novel imaging device to search for residual fluorescence. We determined correlations between tissue fluorescence and histopathology, cathepsin protease expression, and development of recurrent disease. Minimum followup was 9 months (mean, 12 months; range, 9-15 months). RESULTS: Fluorescence was apparent from all 10 tumors and ranged from 3 × 10(7) to 1 × 10(9) counts/millisecond/cm(2). During intraoperative imaging, normal skeletal muscle showed no residual fluorescence. Histopathologic assessment of surgical margins correlated with intraoperative imaging in nine of 10 cases; in the other case, there was no residual fluorescence, but tumor was found at the margin on histologic examination. No animals had recurrent disease at 9 to 15 months. CONCLUSIONS: These initial findings suggest this imaging system might be useful to intraoperatively detect residual tumor after wide resections. CLINICAL RELEVANCE: The ability to assess the tumor bed intraoperatively for residual disease has the potential to improve local control.