PD-L1ATTAC mice reveal the potential of depleting PD-L1 expressing cells in cancer therapy.

Antibodies targeting the PD-1 receptor and its ligand PD-L1 have shown impressive responses in some tumors of bad prognosis. We hypothesized that, since immunosuppressive cells might present several immune checkpoints on their surface, the selective elimination of PD-L1 expressing cells could be efficacious in enabling the activation of antitumoral immune responses. To address this question, we developed an inducible suicidal knock-in mouse allele of Pd-l1 (PD-L1ATTAC) which allows for the tracking and specific elimination of PD-L1-expressing cells in adult tissues. Consistent with our hypothesis, elimination of PD-L1 expressing cells from the mouse peritoneum increased the septic response to lipopolysaccharide (LPS), due to an exacerbated inflammatory response to the endotoxin. In addition, mice depleted of PD-L1+ cells were resistant to colon cancer peritoneal allografts, which was associated with a loss of immunosuppressive B cells and macrophages, concomitant with an increase in activated cytotoxic CD8 T cells. Collectively, these results illustrate the usefulness of PD-L1ATTAC mice for research in immunotherapy and provide genetic support to the concept of targeting PD-L1 expressing cells in cancer.

Activation of the integrated stress response is a vulnerability for multidrug-resistant FBXW7-deficient cells.

FBXW7 is one of the most frequently mutated tumor suppressors, deficiency of which has been associated with resistance to some anticancer therapies. Through bioinformatics and genome-wide CRISPR screens, we here reveal that FBXW7 deficiency leads to multidrug resistance (MDR). Proteomic analyses found an upregulation of mitochondrial factors as a hallmark of FBXW7 deficiency, which has been previously linked to chemotherapy resistance. Despite this increased expression of mitochondrial factors, functional analyses revealed that mitochondria are under stress, and genetic or chemical targeting of mitochondria is preferentially toxic for FBXW7-deficient cells. Mechanistically, the toxicity of therapies targeting mitochondrial translation such as the antibiotic tigecycline relates to the activation of the integrated stress response (ISR) in a GCN2 kinase-dependent manner. Furthermore, the discovery of additional drugs that are toxic for FBXW7-deficient cells showed that all of them unexpectedly activate a GCN2-dependent ISR regardless of their accepted mechanism of action. Our study reveals that while one of the most frequent mutations in cancer reduces the sensitivity to the vast majority of available therapies, it renders cells vulnerable to ISR-activating drugs.

Scaling Up the Family Integrated Care Model in a Level IIIC Neonatal Intensive Care Unit: A Systematic Approach to the Methods and Effort Taken for Implementation.

Background: Family Integrated Care (FICare) integrates parents in the direct care of their child while the healthcare personnel act as teachers and guides. To this date, most reports on the feasibility of this model refer to stable preterm infants admitted to Neonatal Intensive Care Units (NICUs). Objectives: To scale up and adapt FICare to make it suitable in level IIIC NICUs, which care for extreme prematurity and other complex medical or surgical neonatal conditions. Materials and Methods: Step 1 was the creation of the FICare implementation team (FICare-IT) and baseline analysis of current procedures for critical care to identify needs, wishes, and requirements; we aimed for protocol elaboration tailored to our cultural, architectural, and clinical context (March 2017 to April 2018). Step 2 as a dissemination strategy by FICare-IT acting as primary trainers and mentors to ensure the education of 90% of nursing staff (May 2018 to July 2018). Step 3 involved piloting and evaluation with the aim to refine the procedure (July 2018 to December 2020). Results: A rigorous but flexible protocol was edited. The FICare educational manual included two curricula: for healthcare professionals/staff (Training the trainers) and for families (Education of caregivers), the latter being categorized in two intervention levels (basic and advanced), depending on the infant care needs and parent's decision. In total, 76 families and 91 infants (74.7% preterm; 18.7% complex surgery; 6.6% others) were enrolled in the pilot. No differences in acceptance rate (overall 86.4%) or in the number of infant-family dyads in the program per month were observed when considering the pre- and post-Covid-19 pandemic periods. All families, except for one who dropped out of the program, completed the agreed individualized training. Mothers spent more time in NICU than fathers (p < 0.05); uninterrupted time spent by mothers in NICU was longer during the pre-pandemic period (p < 0.01). Observed time to reach proficiency by task was within the expected time in 70% of the program contents. The parents revealed educational manuals, workshops, and cot-side teaching sessions as essential for their training, and 100% said they would accept entry into the FICare program again. Conclusions: The principles of the FICare model are suitable for all levels of care in NICUs. Leadership and continuous evaluation/refinement of implementation procedures are essential components to achieve the objectives.

[20 years urethroplasty experience. Retrospective review and outcomes.] / 20 años realizando uretroplastias. Revisión retrospectiva y resultados.

OBJECTIVE: To present the evolution in the diagnosis and treatment of urethral stricture, after performing 300 surgical procedures over urethral meatus,penile and bulbar urethra along 20 years, contrasting two surgical periods: from 1997-2006 to 2007-2016. MATERIAL AND METHODS: A retrospective review of495 medical records between 1997-2016 was conducted.All the patients treated with urethroplasty were included and those who under went internal urethrotomy,stents or dilatations plus those with strictures due to prostate cancer treatment or orthotopic neobladder were excluded. RESULTS: 300 patients were selected: 100 patients within the first period (1997-2006) and 200 within the second (2007-2016). The median follow-up was 36 months (range 12-60). In relation to the surgical techniques, among the most employed, four are outstanding so their results can be compared in both periods:termino-terminal urethroplasty, penile flap urethroplasty and the buccal mucosa in penile or bulbar urethroplasty.Other techniques were incorporated during the second period. The best outcomes were provided by end' to endurethroplasty with 90 and 92% success. Over the second period, buccal mucosa indications were consolidated with an increase use from 16% to 56%. Were considered as successful those patients that did not need any endoscopic procedure and reporting excellent urinary flow without low urinary tract symptoms. CONCLUSIONS: A trend towards an increased usage of open surgery vs urethrotomy is observed. Buccal mucosa graft has been consolidated as a reconstructive technique. End-to end urethroplasty seems to provide the best functional outcomes.

OBJETIVO: Mostrar la evolución en el diagnóstico y tratamiento de la estenosis uretral tras realizar 300 procedimientos quirúrgicos sobre meato uretral, uretra peneana y bulbar a lo largo de veinte años, comparando dos períodos: 1997-2006 y 2007-2016. MATERIAL Y METODOS: Llevamos a cabo una revisión de los historiales de 495 pacientes diagnosticados de estenosis de uretra entre 1997 y 2016. Incluímos todos aquellos que fueron sometidos a algún tipo de uretroplastia y fueron excluidos los tratados mediante uretrotomía interna, stents, dilataciones, y aquellos que presentaban estenosis a nivel de anastomosis, secundarias a tratamiento del cáncer de próstata o realización de vejigas ortotópicas. RESULTADOS: Seleccionamos 300 pacientes, 100 en el primer período (1997-2006) y 200 en el segundo (2007-2016). La media de seguimiento fue de 36 meses (12-60). Entre las técnicas empleadas destacamos cuatro de ellas para comparar los resultados en ambos períodos: Uretroplastia término terminal, Uretroplastia con mucosa oral en uretra peneana y bulbar y Uretroplastia con colgajo en uretra peneana. En el grupo 1,de 100 pacientes obtuvimos buenos resultados en el 83% y en el grupo 2, de 200 pacientes en el 81%.La técnica que mejores resultados proporcionó fue la término terminal con un 90 y 92% respectivamente. Enel segundo período se consolidó el uso de mucosa oral pasando de utilizarla en un 16% de los casos a un 56%. Consideramos buen resultado funcional aquellos pacientes que no precisaron ningún tipo de manipulación endoscópica o quirúrgica y manifestaron confort miccional con ausencia de síntomas obstructivos o irritativos. CONCLUSIONES: Aumenta el número de pacientes con estenosis de uretra tratados mediante cirugía abierta frente a la uretrotomía. Se consolida el empleo de la mucosa oral en todas sus variantes y se confirma que la uretroplastia término terminal es la técnica que proporciona mejores resultados.

20 años realizando uretroplastias. Revisión retrospectiva y resultados / 20 years urethroplasty experience. Retrospective review and outcomes

OBJETIVO: Mostrar la evolución en el diagnóstico y tratamiento de la estenosis uretral tras realizar 300 procedimientos quirúrgicos sobre meato uretral, uretra peneana y bulbar a lo largo de veinte años, comparando dos períodos: 1997-2006 y 2007-2016. MATERIAL Y METODOS: Llevamos a cabo una revisión de los historiales de 495 pacientes diagnosticados de estenosis de uretra entre 1997 y 2016. Incluímos todos aquellos que fueron sometidos a algún tipo de uretroplastia y fueron excluidos los tratados mediante uretrotomía interna, stents, dilataciones, y aquellos que presentaban estenosis a nivel de anastomosis, secundarias a tratamiento del cáncer de próstata o realización de vejigas ortotópicas. RESULTADOS: Seleccionamos 300 pacientes, 100 en el primer período (1997-2006) y 200 en el segundo (2007-2016). La media de seguimiento fue de 36 meses (12-60). Entre las técnicas empleadas destacamos cuatro de ellas para comparar los resultados en ambos períodos: Uretroplastia término terminal, Uretroplastia con mucosa oral en uretra peneana y bulbar y Uretroplastia con colgajo en uretra peneana. En el grupo 1, de 100 pacientes obtuvimos buenos resultados en el 83% y en el grupo 2, de 200 pacientes en el 81%.La técnica que mejores resultados proporcionó fue la término terminal con un 90 y 92% respectivamente. Enel segundo período se consolidó el uso de mucosa oral pasando de utilizarla en un 16% de los casos a un 56%. Consideramos buen resultado funcional aquellos pacientes que no precisaron ningún tipo de manipulación endoscópica o quirúrgica y manifestaron confort miccional con ausencia de síntomas obstructivos o irritativos. CONCLUSIONES: Aumenta el número de pacientes con estenosis de uretra tratados mediante cirugía abierta frente a la uretrotomía. Se consolida el empleo de la mucosa oral en todas sus variantes y se confirma que la uretroplastia término terminal es la técnica que proporciona mejores resultados

OBJECTIVE: To present the evolution in the diagnosis and treatment of urethral stricture, after performing 300 surgical procedures over urethral meatus,penile and bulbar urethra along 20 years, contrasting two surgical periods: from 1997-2006 to 2007-2016. MATERIAL AND METHODS: A retrospective review of495 medical records between 1997-2016 was conducted.All the patients treated with urethroplasty were included and those who under went internal urethrotomy,stents or dilatations plus those with strictures due to prostate cancer treatment or orthotopic neobladder were excluded. RESULTS: 300 patients were selected: 100 patients within the first period (1997-2006) and 200 within the second (2007-2016). The median follow-up was 36 months (range 12-60). In relation to the surgical techniques, among the most employed, four are outstanding so their results can be compared in both periods:termino-terminal urethroplasty, penile flap urethroplasty and the buccal mucosa in penile or bulbar urethroplasty.Other techniques were incorporated during the second period. The best outcomes were provided by end’ to endurethroplasty with 90 and 92% success. Over the second period, buccal mucosa indications were consolidated with an increase use from 16% to 56%. Were considered as successful those patients that did not need any endoscopic procedure and reporting excellent urinary flow without low urinary tract symptoms. CONCLUSIONS: A trend towards an increased usage of open surgery vs urethrotomy is observed. Buccal mucosa graft has been consolidated as a reconstructive technique. End-to end urethroplasty seems to provide the best functional outcomes

ImmunoPEGliposome-mediated reduction of blood and brain amyloid levels in a mouse model of Alzheimer's disease is restricted to aged animals.

The accumulation of extracellular amyloid-beta (Aß) and intracellular neurofibrillary tangles (hyper-phosphorylated Tau) in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). Active and passive immunotherapy may limit cerebral Aß deposition and/or accelerate its clearance. With the aid of a newly characterized monoclonal anti-Aß antibody we constructed immunoPEGliposomes with high avidity for capturing Aß in the periphery. The functionality of these vesicles in modulating Aß uptake by both human brain capillary endothelial hCMEC/D3 cells (suppressing uptake) and THP-1 phagocytes (stimulating uptake) was confirmed in vitro. The multivalent immunoliposomes dramatically reduced circulating and brain levels of Aß1-40, and particularly Aß1-42, in "aged" (16 month-old), but not "adult" (10 month-old) APP/PS1 transgenic mice on repeated intraperitoneal administration. Furthermore, the immunoPEGliposome-mediated reduction in amyloidosis correlated with lower levels of glial fibrillary acidic protein (GFAP) and reactive glia (GFAP-positive cells). This treatment also lowered the ratio of phosphorylated Tau to total Tau. The therapeutic efficacy of immunoliposome treatment was superior to free monoclonal antibody administration (at an equivalent antibody dose). The potential mechanisms and significance of age-dependent immunoliposome therapy in AD is discussed.

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML.

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.

AßPP/PS1 Transgenic Mice Show Sex Differences in the Cerebellum Associated with Aging.

Cerebellar pathology has been related to presenilin 1 mutations in certain pedigrees of familial Alzheimer's disease. However, cerebellum tissue has not been intensively analyzed in transgenic models of mutant presenilins. Furthermore, the effect of the sex of the mice was not systematically analyzed, despite the fact that important gender differences in the evolution of the disease in the human population have been described. We analyzed whether the progression of amyloidosis in a double transgenic mouse, AßPP/PS1, is susceptible to aging and differentially affects males and females. The accumulation of amyloid in the cerebellum differentially affects males and females of the AßPP/PS1 transgenic line, which was found to be ten-fold higher in 15-month-old females. Amyloid-ß accumulation was more evident in the molecular layer of the cerebellum, but glia reaction was only observed in the granular layer of the older mice. The sex divergence was also observed in other neuronal, survival, and autophagic markers. The cerebellum plays an important role in the evolution of the pathology in this transgenic mouse model. Sex differences could be crucial for a complete understanding of this disease. We propose that the human population could be studied in this way. Sex-specific treatment strategies in human populations could show a differential response to the therapeutic approach.

POLD3 Is Haploinsufficient for DNA Replication in Mice.

The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologs are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizosaccharomyces pombe ortholog is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals. Strikingly, even Pold3(+/-) mice were born at sub-Mendelian ratios, and, of those born, some presented hydrocephaly and had a reduced lifespan. In cells, POLD3 deficiency led to replication stress and cell death, which were aggravated by the expression of activated oncogenes. Finally, we show that Pold3 deletion destabilizes all members of the Polδ complex, explaining its major role in DNA replication and the severe impact of its deficiency.

Peripheral amyloid levels present gender differences associated with aging in AßPP/PS1 mice.

The accumulation of amyloid-ß (Aß) peptide is one of the major neuropathological hallmarks of Alzheimer's disease (AD). We have analyzed whether the progression of amyloidosis differentially affects males and females along aging in AßPP/PS1 transgenic mice. The levels of peripheral amyloid, Aß40 and Aß42, are not modified in either sex until 9 months of age. After that, however, there is an increase in amyloid levels in plasma among females and a decrease among males. These findings could be essential to design gender-specific strategies in other in vivo experiments or even in AD treatments.

Repeated intraperitoneal injections of liposomes containing phosphatidic acid and cardiolipin reduce amyloid-ß levels in APP/PS1 transgenic mice.

The accumulation of extracellular amyloid-beta (Aß) peptide and intracellular neurofibrillary tangles in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). It is thought that an equilibrium exists between Aß in the brain and in the peripheral blood and thus, it was hypothesized that shifting this equilibrium towards the blood by enhancing peripheral clearance might reduce Aß levels in the brain: the 'sink effect'. We tested this hypothesis by intraperitoneally injecting APP/PS1 transgenic mice with small unilamellar vesicles containing either phosphatidic acid or cardiolipin over 3weeks. This treatment reduced significantly the amount of Aß in the plasma and the brain levels of Aß were lighter affected. Nevertheless, this dosing regimen did modulate tau phosphorylation and glycogen synthase kinase 3 activities in the brain, suggesting that the targeting of circulating Aß may be therapeutically relevant in AD. FROM THE CLINICAL EDITOR: Intraperitoneal injection of small unilamellar vesicles containing phosphatidic acid or cardiolipin significantly reduced the amount of amyloid-beta (Aß) peptide in the plasma in a rodent model. Brain levels of Aß were also affected - although to a lesser extent - suggesting that targeting of circulating Aß may be therapeutically relevant of Alzheimer's disease.

Cellular prion protein modulates ß-amyloid deposition in aged APP/PS1 transgenic mice.

Alzheimer's disease and prion diseases are neuropathological disorders that are caused by abnormal processing and aggregation of amyloid and prion proteins. Interactions between amyloid precursor protein (APP) and PrP(c) proteins have been described at the neuron level. Accordingly to this putative interaction, we investigated whether ß-amyloid accumulation may affect prion infectivity and, conversely, whether different amounts of PrP may affect ß-amyloid accumulation. For this purpose, we used the APPswe/PS1dE9 mouse line, a common model of Alzheimer's disease, crossed with mice that either overexpress (Tga20) or that lack prion protein (knock-out) to generate mice that express varying amounts of prion protein and deposit ß-amyloid. On these mouse lines, we investigated the influence of each protein on the evolution of both diseases. Our results indicated that although the presence of APP/PS1 and ß-amyloid accumulation had no effect on prion infectivity, the accumulation of ß-amyloid deposits was dependent on PrP(c), whereby increasing levels of prion protein were accompanied by a significant increase in ß-amyloid aggregation associated with aging.

Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia.

BACKGROUND: Estradiol has been shown to exert neuroprotective effects in several neurodegenerative conditions, including cerebral ischemia. The presence of this hormone prior to ischemia attenuates the damage associated with such events in a rodent model (middle cerebral artery occlusion (MCAO)), although its therapeutic value when administered post-ischemia has not been assessed. Hence, we evaluated the effects of estradiol treatment after permanent MCAO (pMCAO) was induced in rats, studying the PI3K/AKT/GSK3/ß-catenin survival pathway and the activation of SAPK-JNK in two brain areas differently affected by pMCAO: the cortex and hippocampus. In addition, we analyzed the effect of estradiol on the glial response to injury. METHODS: Male rats were subjected to pMCAO and estradiol (0.04 mg/kg) was administered 6, 24, and 48 h after surgery. The animals were sacrificed 6 h after the last treatment, and brain damage was evaluated by immunohistochemical quantification of 'reactive gliosis' using antibodies against GFAP and Iba1. In addition, Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), GSK3, phospho-GSK3(Ser21/9), ß-catenin, SAPK-JNK, and pSAPK-JNK(Thr183/Tyr185) levels were determined in western blots of the ipsilateral cerebral cortex and hippocampus, and regional differences in neuronal phospho-Akt expression were determined by immunohistochemistry. RESULTS: The increases in the percentage of GFAP- (5.25-fold) and Iba1- (1.8-fold) labeled cells in the cortex and hippocampus indicate that pMCAO induced 'reactive gliosis'. This effect was prevented by post-ischemic estradiol treatment; diminished the number of these cells to those comparable with control animals. pMCAO down-regulated the PI3K/AkT/GSK3/ß-catenin survival pathway to different extents in the cortex and hippocampus, the activity of which was restored by estradiol treatment more efficiently in the cerebral cortex (the most affected region) than in the hippocampus. No changes in the phosphorylation of SAPK-JNK were observed 54 h after inducing pMCAO, whereas pMCAO did significantly decrease the phospho-Akt(Ser473) in neurons, an effect that was reversed by estradiol. CONCLUSION: The present study demonstrates that post-pMCAO estradiol treatment attenuates ischemic injury in both neurons and glia, events in which the PI3K/AKT/GSK3/ß-catenin pathway is at least partly involved. These findings indicate that estradiol is a potentially useful treatment to enhance recovery after human ischemic stroke.

Listening to patients' voices: linguistic indicators related to diabetes self-management.

A great deal of research in health care has examined a wide range of variables to better understand the degree to which patients follow the advice of medical professionals in managing their health, known as adherence. This paper explains the development of the linguistic systems to describe and evaluate two psychosocial constructs (i.e. control orientation and agency) that have been found to be related to adherence in previous research for subjects with diabetes (Trento et al. 2007; Wangberg 2007; O'Hea et al. 2009). The present data came from 43 semi-structured in-depth interviews of subjects with Type 2 diabetes. One-on-one interviews with open-ended questions elicited subjects' 'stories' about living with diabetes, and the transcribed interviews were analyzed to develop the linguistic systems of control orientation and agency. The resultant systems were applied to the 43 interviews by raters with high inter-rater reliability. The results showed demarcations of clearly identified codings of patient types. The paper presents the linguistic coding systems developed in the study, the results of their application to the patient interview data, and recommendations for improved communication with patients.