Photo-Cross-linked Gelatin Methacryloyl Hydrogels Enable the Growth of Primary Human Endometrial Stromal Cells and Epithelial Gland Organoids.

In vitro three-dimensional (3D) models are better able to replicate the complexity of real organs and tissues than 2D monolayer models. The human endometrium, the inner lining of the uterus, undergoes complex changes during the menstrual cycle and pregnancy. These changes occur in response to steroid hormone fluctuations and elicit crosstalk between the epithelial and stromal cell compartments, and dysregulations are associated with a variety of pregnancy disorders. Despite the importance of the endometrium in embryo implantation and pregnancy establishment, there is a lack of in vitro models that recapitulate tissue structure and function and as such a growing demand for extracellular matrix hydrogels that can support 3D cell culture. To be physiologically relevant, an in vitro model requires mechanical and biochemical cues that mimic those of the ECM found in the native tissue. We report a semisynthetic gelatin methacryloyl (GelMA) hydrogel that combines the bioactive properties of natural hydrogels with the tunability and reproducibility of synthetic materials. We then describe a simple protocol whereby cells can quickly be encapsulated in GelMA hydrogels. We investigate the suitability of GelMA hydrogel to support the development of an endometrial model by culturing the main endometrial cell types: stromal cells and epithelial cells. We also demonstrate how the mechanical and biochemical properties of GelMA hydrogels can be tailored to support the growth and maintenance of epithelial gland organoids that emerge upon 3D culturing of primary endometrial epithelial progenitor cells in a defined chemical medium. We furthermore demonstrate the ability of GelMA hydrogels to support the viability of stromal cells and their function measured by monitoring decidualization in response to steroid hormones. This study describes the first steps toward the development of a hydrogel matrix-based model that recapitulates the structure and function of the native endometrium and could support applications in understanding reproductive failure.

Gender Differences in Experiences and Expectations of Hemodialysis in a Frail and Seriously Unwell Patient Population.

Introduction: Surprisingly few studies have explored the experiences of seriously unwell people with kidney disease on hemodialysis therapy: we conducted a mixed-methods study to investigate gender differences in illness experience, symptom burden, treatment considerations or expectations in this cohort. Methods: Seriously unwell people on hemodialysis (1-year mortality risk of >20%) at 3 hospital-based units were invited to take part in a structured interview or to complete the same questions independently via a questionnaire. A total of 54 people took part (36 males, 18 females); data analysis was undertaken using a thematic approach. Results: "Desire to keep living" is the most important and basic thought process when starting dialysis. Fear also predominates influencing risk assessment and decision-making. Once fear is managed, there are physical, social, practical and emotional issues to rationalize, but choice only seems possible if shared decision-making is part of the consultation.Gender differences were seen in perceived hopes and expectations of treatment. Males were more likely to prioritize achievement of physical goals, with females prioritizing a wish to feel well. Both genders reported significantly higher symptom scores than their health care provider perceived, however this difference was more marked in females. Dialysis regret existed in >50% of participants and 6 out of 54 (11%) stated that they would have chosen no dialysis at all. Females were more likely to report feeling depressed (P = 0.001). Conclusion: Different genders approach treatment decisions and prioritize treatment expectations differently. Recognizing this will allow personalized care plans to be developed and improve the experiences of seriously unwell people with kidney disease.

Long term central venous catheters for haemodialysis causing an extra-cardiac right-to-left shunt.

Central venous catheters can provide long-term access for haemodialysis patients who cannot have an arteriovenous fistula or graft fashioned. However, long-term central venous catheter use for haemodialysis may lead to complications including central venous stenosis, and superior vena cava obstruction in its worst form. Here, we describe the case of a patient on haemodialysis via central venous catheters for over 20 years, in whom chronic superior vena cava obstruction led to the development of mediastinal collateral vessels. These drained deoxygenated systemic venous blood into the oxygenated pulmonary venous system. Over time, this caused a significant right-to-left shunt and resulting hypoxaemia. This is the first reported case of central venous catheters used for haemodialysis resulting in an acquired, extra-cardiac, right-to-left shunt.

Lithium use in a patient on haemodialysis with bipolar affective disorder and lithium-induced nephropathy.

Lithium is an effective mood stabiliser used to treat bipolar affective disorder (BPAD); however, it can also adversely affect the kidneys, causing acute toxic effects, nephrogenic diabetes insipidus, chronic renal dysfunction and end-stage kidney disease (ESKD) in a minority of patients. We describe the case of a man with a 34-year history of BPAD type-1 and a 2-year history of ESKD secondary to lithium-induced nephropathy who experienced a manic relapse. He previously responded well to lithium but, following a deterioration in kidney function, was switched to olanzapine and sodium valproate. This precipitated a period of instability, which culminated in a treatment-resistant manic episode requiring hospital admission. After a multidisciplinary team discussion, lithium therapy was restarted and provided remission. This was achieved safely through a reduced dosing schedule of three times a week post dialysis, slow dose titration and blood level monitoring prior to each dialysis session.

Perceptions of Illness Severity, Treatment Goals, and Life Expectancy: The ePISTLE Study.

INTRODUCTION: A better understanding of factors influencing perceived life expectancy (PLE), interactions between patient prognostic beliefs, experiences of illness, and treatment behavior is urgently needed. METHODS: Case-notes at 3 hemodialysis units were screened: patients with ≥20% 1-year mortality risk were included. Patients and their health care professionals (HCPs) were invited to complete a structured interview or mixed-methods questionnaire. Four hundred eleven patient notes were screened. Seventy-seven eligible patients were approached and 51 were included. RESULTS: Patients predicted significantly higher life expectancies than HCPs (P < 0.0001). Documented cognitive impairment, gender, or increasing age did not affect 1- or 5-year PLE. PLE influenced priorities of care: one-fifth of patients who estimated themselves to have >95% 1-year survival preferred "care focusing on relieving pain and discomfort," compared with nearly three-quarters of those reporting a ≤50% chance of 1-year survival. Twenty of 51 (39%) patients believed transplantation was an option for them, despite only 4 being waitlisted at the time of the interview. Patients who thought they were transplant candidates were significantly more confident they would be alive at 1 and 5 years and to want resuscitation attempted. Cognitive impairment had no effect on perceived transplant candidacy. A high symptom burden was present and underrecognized by HCPs. High symptom burden was associated with significantly lower PLE at both 1 and 5 years, increased anxiety/depression scores, and treatment choices more likely to prioritize relief of suffering. CONCLUSION: There is a disparity between patient PLE and those of their HCPs. Severity of symptom burden and beliefs regarding PLE or transplant candidacy affect patient treatment preferences.

Embryo biosensing by uterine natural killer cells determines endometrial fate decisions at implantation.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation-like disposal of low-fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low-quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro-inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high molecular weight HA (HMWHA) inhibits uNK cell-mediated killing of senescent decidual cells. In contrast, low molecular weight HA (LMWHA) did not attenuate uNK cell activity in co-culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo-mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation.

N-Ethyl-N-nitrosourea mutagenesis in the mouse provides strong genetic and in vivo evidence for the role of the Caspase Recruitment Domain (CARD) of CARD-MAGUK1 in T regulatory cell development.

Natural regulatory T (nTreg) cells generated in the thymus are essential throughout life for the maintenance of T-cell homeostasis and the prevention of autoimmunity. T-cell receptor (TCR)/CD28-mediated activation of nuclear factor-κB and (J)un (N)-terminal kinase pathways is known to play a key role in nTreg cell development but many of the predicted molecular interactions are based on extrapolations from non-Treg cell TCR stimulation with non-physiological ligands. For the first time, we provide strong genetic evidence of a scaffold function for the Caspase Recruitment Domain (CARD) of the TCR signalling protein CARD-MAGUK1 (CARMA1) in nTreg cell development in vivo. We report two, new, N-ethyl-N-nitrosourea-derived mutant mice, Vulpo and Zerda, with a profound block in the development of nTreg cells in the thymus as well as impaired inducible Treg cell differentiation in the periphery. Despite independent heritage, both mutants harbour different point mutations in the CARD of the CARMA1 protein. Mutations in vulpo and zerda do not affect expression levels of CARMA1 but still impair signalling through the TCR due to defective downstream Bcl-10 recruitment by the mutated CARD of CARMA1. Phenotypic differences observed between Vulpo and Zerda mutants suggest a role for the CARD of CARMA1 independent of Bcl-10 activation of downstream pathways. We conclude that our forward genetic approach demonstrates a critical role for the CARD function of CARMA1 in Treg cell development in vivo.

Transplantation tolerance.

Although transplantation has been a standard medical practice for decades, marked morbidity from the use of immunosuppressive drugs and poor long-term graft survival remain important limitations in the field. Since the first solid organ transplant between the Herrick twins in 1954, transplantation immunology has sought to move away from harmful, broad-spectrum immunosuppressive regimens that carry with them the long-term risk of potentially life-threatening opportunistic infections, cardiovascular disease, and malignancy, as well as graft toxicity and loss, towards tolerogenic strategies that promote long-term graft survival. Reports of "transplant tolerance" in kidney and liver allograft recipients whose immunosuppressive drugs were discontinued for medical or non-compliant reasons, together with results from experimental models of transplantation, provide the proof-of-principle that achieving tolerance in organ transplantation is fundamentally possible. However, translating the reconstitution of immune tolerance into the clinical setting is a daunting challenge fraught with the complexities of multiple interacting mechanisms overlaid on a background of variation in disease. In this article, we explore the basic science underlying mechanisms of tolerance and review the latest clinical advances in the quest for transplantation tolerance.

Measuring the quality of end of life management in patients with advanced kidney disease: results from the pan-Thames renal audit group.

BACKGROUND: Despite a recent increased awareness of the need for quality End of Life (EOL) care for patients with advanced kidney disease, there is no established method for measuring or auditing outcomes relating to EOL care in this population. METHODS: We designed a one-page proforma, which was used to collect data on various aspects of EOL care relating to all deaths of patients on dialysis and patients dying on specialist renal wards, over a predefined 8-week period in 10 hospitals in London and South-East England. RESULTS: One hundred and thirty-eight deaths were recorded over the 8-week study period. The majority of patients (83%) were receiving maintenance haemodialysis prior to their terminal presentation. About 69% of deaths occurred during an in-patient hospital admission-of these, 36% were considered 'unexpected' and most quality markers of good EOL management were significantly less likely to be achieved in these patients, including use of palliative care strategies, good symptom control and overall quality of death. Thirty-six per cent of patients were from various ethnic minorities, and in this group, there was a trend towards lower use of palliative care pathways and lower rates of withdrawal from dialysis. CONCLUSIONS: This study confirms that it is possible to measure many important outcomes relating to quality of EOL care using a proforma completed at the time of death. Our findings suggest that many aspects of good EOL care are under-achieved in our region. This, in part, is due to a failure to recognize the worsening trajectory of the deteriorating patient, resulting in missed opportunities for EOL care planning and appropriate symptom control. Our observations suggest that there is a need for improved education and training in this area, particularly in detection of the dying patient, the value of advance care planning and the utility of tools such as the Liverpool Care Pathway.

Renal transplantation in systemic vasculitis: when is it safe?

BACKGROUND: There are no clear guidelines on renal transplantation in patients with antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis. METHODS: We undertook a survey of transplant centres across Europe to assess whether there was consensus about how to manage transplantation in patients with vasculitis. We then identified 107 renal allograft recipients whose primary disease was systemic vasculitis and assessed their outcome post-transplant. RESULTS: All questionnaire respondents felt that vasculitis should be in remission at transplantation, 16% believed that ANCA should be negative pre-transplant and 40% felt that one should wait >12 months after remission before transplanting. Remission was defined by all as an absence of clinical symptoms of vasculitis, but three respondents (13%) also required a negative ANCA test. Overall graft survival was 70% after 10 years (95% C.I. 58-82). A total of 30 (41% of those with known ANCA status) were ANCA-positive peri-transplantation, while 15 (14%) were transplanted <1 year post-remission. Severe vasculopathy occurred more frequently in ANCA-positive recipients (odds ratio 4.4, 95% C.I. 1.1-16.8, P < 0.05), although causation cannot be determined from this study. Vasculopathy significantly reduced 10-year graft survival to 47% (P < 0.05). However, ANCA status per se was not significantly associated with graft failure. The strongest predictor of death was transplantation <1 year post-vasculitis remission on both univariate and multivariate analysis (hazard ratio 2.3, P < 0.05). CONCLUSIONS: In conclusion, circulating ANCA at transplant was associated with the development of vascular lesions in the graft but was not significantly correlated with graft survival. Most grafts were lost due to patient death, which was more likely if transplantation occurred <12 months following induction of remission of ANCA-positive vasculitis.