Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles.

Purpose: Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs). Patients and Methods: Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals' urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry. Results: The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side. Conclusion: The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.

Antenatal Ureaplasma Infection Causes Colonic Mucus Barrier Defects: Implications for Intestinal Pathologies.

Chorioamnionitis is a risk factor for necrotizing enterocolitis (NEC). Ureaplasma parvum (UP) is clinically the most isolated microorganism in chorioamnionitis, but its pathogenicity remains debated. Chorioamnionitis is associated with ileal barrier changes, but colonic barrier alterations, including those of the mucus barrier, remain under-investigated, despite their importance in NEC pathophysiology. Therefore, in this study, the hypothesis that antenatal UP exposure disturbs colonic mucus barrier integrity, thereby potentially contributing to NEC pathogenesis, was investigated. In an established ovine chorioamnionitis model, lambs were intra-amniotically exposed to UP or saline for 7 d from 122 to 129 d gestational age. Thereafter, colonic mucus layer thickness and functional integrity, underlying mechanisms, including endoplasmic reticulum (ER) stress and redox status, and cellular morphology by transmission electron microscopy were studied. The clinical significance of the experimental findings was verified by examining colon samples from NEC patients and controls. UP-exposed lambs have a thicker but dysfunctional colonic mucus layer in which bacteria-sized beads reach the intestinal epithelium, indicating undesired bacterial contact with the epithelium. This is paralleled by disturbed goblet cell MUC2 folding, pro-apoptotic ER stress and signs of mitochondrial dysfunction in the colonic epithelium. Importantly, the colonic epithelium from human NEC patients showed comparable mitochondrial aberrations, indicating that NEC-associated intestinal barrier injury already occurs during chorioamnionitis. This study underlines the pathogenic potential of UP during pregnancy; it demonstrates that antenatal UP infection leads to severe colonic mucus barrier deficits, providing a mechanistic link between antenatal infections and postnatal NEC development.

Genomic and epidemiological insight of an outbreak of carbapenemase-producing Enterobacterales in a Portuguese hospital with the emergence of the new KPC-124.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) is an increasing problem in healthcare settings. This study aimed to identify the source of a CPE outbreak that occurred in 2022, in a tertiary hospital in the North of Portugal, to identify exposed patients, and to assess the risk of becoming CPE-positive following hospital admission. METHODS: A multi-disciplinary investigation was conducted including descriptive, analytical, and molecular epidemiology, environmental screening, and assessment of infection control measures. Clinical and environmental isolates were analyzed using whole-genome sequencing and phylogenetic analysis. Additionally, a prospective observational cohort study was conducted to further investigate the risk factors associated with the emergence of new cases in cohorts of CPE-negative admitted patients. RESULTS: We observed the presence of multispecies KPC-, IMP-, and/or NDM-producing isolates. Genetically indistinguishable clinical and environmental isolates were found on the same room/ward. The ST45 KPC-3-producing Klebsiella pneumoniae clone was the responsible for the outbreak. During patients' treatment, we detected the emergence of resistance to ceftazidime-avibactam, associated with mutations in the blaKPC-3 gene (blaKPC-46, blaKPC-66 and blaKPC-124, the last variant never previously reported), suggesting a vertical evolutionary trajectory. Patients aged ≥ 75 years, hygiene/feeding-care dependent, and/or subjected to secretion aspiration were risk factors for CPE colonization after hospital admission. Additionally, cases with previous admission to the emergency department suggest that CPE dissemination may occur not only during hospitalization but also in the emergency department. CONCLUSION: Overall, the study highlights that selection pressure with antibiotics, like ceftazidime-avibactam, is a contributing factor to the emergence of new ß-lactamase variants and antibiotic resistance. It also shows that the hospital environment can be a significant source of CPE transmission, and that routine use of infection control measures and real-time molecular epidemiology investigations are essential to ensure the long-term termination of CPE outbreaks and prevent future resurgences.

Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Local Flaps to Cover Skin Necrosis after Skin-sparing Mastectomy and Prepectoral Reconstruction from PreQ-20 Trial.

Background: In recent years, mastectomy has increasingly been indicated for women at high risk and those with breast cancer. Prepectoral reconstruction with polyurethane implant is an option for these patients. Nevertheless, this procedure can become complicated with exposure of the implant. The aim of this article is to describe the feasibility of local flaps to treat skin necrosis and dehiscence after prepectoral reconstruction and its impact on implant loss. Methods: This study includes the women who met the inclusion/exclusion criteria of the PreQ-20 protocol (12), which assessed patients with exposed implant who required a local flap for its coverage. Three types of flaps were used: thoracoepigastric, lateral thoracic, and batwing. Results: The study included 226 skin-sparing mastectomies and immediate reconstruction using prepectoral implants (52.7% bilateral mastectomies). Some 20.9% of the patients showed complications, with wound dehiscence the most frequent. Thirteen local flaps to cover the implant were performed. All flaps presented appropriate perfusion; however, the implant cover failed in six patients (46.2%). Conclusions: The use of local flaps can be a low-morbidity option for preventing implant loss when skin dehiscence or necrosis occurs and delays in oncology treatments.

Jejunal gastrointestinal stromal tumor: a diagnostic challenge.

Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors of the gastrointestinal tract and a rare cause of gastrointestinal bleeding. These tumors usually affect people over 50 years of age and they exhibit a wide range of clinical manifestations, including asymptomatic patients, nonspecific symptoms, obstruction or bleeding, which may delay diagnosis. Early diagnosis and treatment are crucial because GISTs can be aggressive and metastasize. This case highlights the importance of considering GISTs in the differential diagnosis of obscure gastrointestinal bleeding.

Assessment of an intervention for preventing pediatric overweight and obesity through the World Café technique: A qualitative study.

PURPOSE: To evaluate the self-reported perspectives of participants involved in the Previene Cádiz intervention for preventing pediatric overweight and obesity. DESIGN AND METHODS: This qualitative study collected information through the World Café technique. A purposive sample of 40 participants was used, of which 14 were schoolchildren, 12 were teachers, and 14 were parent volunteers. The data were segmented, and concepts were created and grouped into dimensions and categories. RESULTS: The participants confirmed they had learned new information and behaviors about healthy habits. Parental awareness was considered a crucial and necessary element in changing family habits; therefore, increasing the motivation of family members was deemed a critical task in public health interventions conducted in school settings. DISCUSSION: Despite the suitability of qualitative methodology to evaluate the perceptions of the main players in an educational intervention, scientific literature is scarce. Obtaining information from the educational community about an intervention is not always easy, so the perspectives of teachers, students, and families about the Previene Cádiz intervention through the World Café approach is considered a relevant contribution. CONCLUSIONS: The participants considered the intervention positive in terms of learning and fostering increased knowledge, awareness, and healthy behaviors. PRACTICE IMPLICATIONS: Future interventions should encourage the active participation of all social groups involved, integrating dynamic and collaborative training activities that are acceptable to all participants.

An in vitro model system based on calcium- and phosphate ion-induced hMSC spheroid mineralization.

A challenge in regenerative medicine is creating the three-dimensional organic and inorganic in vitro microenvironment of bone, which would allow the study of musculoskeletal disorders and the generation of building blocks for bone regeneration. This study presents a microwell-based platform for creating spheroids of human mesenchymal stromal cells, which are then mineralized using ionic calcium and phosphate supplementation. The resulting mineralized spheroids promote an osteogenic gene expression profile through the influence of the spheroids' biophysical environment and inorganic signaling and require less calcium or phosphate to achieve mineralization compared to a monolayer culture. We found that mineralized spheroids represent an in vitro model for studying small molecule perturbations and extracellular mediated calcification. Furthermore, we demonstrate that understanding pathway signaling elicited by the spheroid environment allows mimicking these pathways in traditional monolayer culture, enabling similar rapid mineralization events. In sum, this study demonstrates the rapid generation and employment of a mineralized cell model system for regenerative medicine applications.

Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.

Our house believes that: The clipped lymph node is the true sentinel node after neoadjuvant chemotherapy in N+ patients.

Axillary staging is an important prognostic factor in breast cancer, being sentinel lymph node biopsy (SLNB) the gold standard staging method in early stages. However, in clinically node positive (cN+) patients who converted to clinically node-negative (cN0) after primary systemic therapy (PST) the axillary staging method during surgery remains controversial. There are at least three validated methods: SLNB, targeted axillary dissection (TAD) and marking axillary nodes with radioactive iodine seeds (MARI) procedure. Our house believe that the biopsied and clipped lymph node could predict response to systemic treatment.

A Lamb1Dendra2 mouse model identifies basement-membrane-producing origins and dynamics in PyMT breast tumors.

The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM.

Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.

Cryogenic electron microscopy and data processing enable the determination of structures of isolated macromolecules to near-atomic resolution. However, these data do not provide structural information in the cellular environment where macromolecules perform their native functions, and vital molecular interactions can be lost during the isolation process. Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET). Cellular cryo-ET benefits from the technological developments in electron microscopes, detectors and data processing, and more in situ structures are being obtained and at increasingly higher resolution. In this Review, we discuss recent studies employing cryo-ET on FIB-generated lamellae and the technological developments in ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing and correlative light and electron microscopy that have enabled these studies. Finally, we explore the future of cryo-ET in terms of both methods development and biological application.

Reconstrucción mamaria inmediata mediante implante prepectoral de poliuretano. Resultados preliminares del estudio prospectivo PreQ-20 / Immediate breast reconstruction by prepectoral polyurethane implant: Preliminary results of the prospective study PreQ-20

Introducción: Durante los últimos años las técnicas de mastectomía y reconstrucción han evolucionado hacia procedimientos menos agresivos, mejorando la satisfacción y calidad de vida de la mujer. Por ello, la mastectomía se ha convertido en una opción válida tanto para mujeres con cáncer de mama como en mujeres de alto riesgo. El objetivo de este estudio es analizar la seguridad de la mastectomía y reconstrucción inmediata prepectoral con implante de poliuretano en mujeres con cáncer de mama y reducción de riesgo. Métodos: Estudio prospectivo observacional para evaluar la factibilidad y seguridad de la reconstrucción inmediata mediante implante prepectoral de poliuretano. Se incluyeron todas las mujeres (con cáncer de mama o alto riesgo para cáncer de mama) intervenidas mediante una mastectomía preservadora de piel o piel y pezón con reconstrucción inmediata con implante de poliuretano prepectoral. Se excluyeron las mujeres con sarcomas de mama, progresión de la enfermedad durante el tratamiento sistémico primario, reconstrucción diferida, autóloga o retropectoral y aquellas pacientes que no desearon participar en el estudio. Los procedimientos quirúrgicos fueron realizados tanto por cirujanos senior como junior. Todas las pacientes recibieron los tratamientos complementarios correspondientes. Se analizaron todos los eventos adversos acontecidos durante el seguimiento y los factores de riesgo para desarrollarlos. Resultados: Se realizaron 159 reconstrucciones en 102 mujeres, el 80,4% por un carcinoma mamario. Catorce pacientes desarrollaron complicaciones, siendo el seroma y la dehiscencia de la herida las más frecuentes. Ocho mujeres precisaron una reintervención (5%), 7 de ellas por exposición del implante. Cuatro reconstrucciones (2,5%) culminaron con pérdida del implante. Tres pacientes presentaron progresión de su proceso oncológico: una recaída local en el colgajo de la mastectomía, una progresión axilar y una progresión sistémica. (AU)

Introduction: In recent years, mastectomy and reconstruction techniques have evolved towards less aggressive procedures, improving the satisfaction and quality of life of women. For this reason, mastectomy has become a valid option for both women with breast cancer and high-risk women. The objective of this study is to analyze the safety of mastectomy and immediate prepectoral reconstruction with polyurethane implant in women with breast cancer and risk reduction. Method: Observational prospective study to evaluate the feasibility and safety of immediate reconstruction using prepectoral polyurethane implant. All women (with breast cancer or high risk for breast cancer) who underwent skin-sparing or skin-and-nipple-sparing mastectomy with immediate reconstruction with a prepectoral polyurethane implant were included. Women with breast sarcomas, disease progression during primary systemic therapy, delayed, autologous or retropectoral reconstruction, and those who did not wish to participate in the study were excluded. Surgical procedures were performed by both senior and junior surgeons. All patients received the corresponding complementary treatments. All adverse events that occurred during follow-up and the risk factors for developing them were analyzed. Results: 159 reconstructions were performed in 102 women, 80.4% due to breast carcinoma. Fourteen patients developed complications, the most frequent being seroma and wound dehiscence. Eight women required a reoperation (5.0%), seven of them due to implant exposure. Four reconstructions (2.5%) resulted in loss of the implant. Three patients progressed from their oncological process: a local relapse in the mastectomy flap, an axillary progression and a systemic progression. (AU)

Safety and Quality of Life in Women with Immediate Reconstruction with Polyurethane Implants after Neoadjuvant Chemotherapy: Outcomes from The Preq-20 Trial.

INTRODUCTION: Various studies have evaluated the impact of neoadjuvant chemotherapy (NAC) on the complications of breast cancer surgery, most of which were retrospective and did not assess the variables related to postoperative risk factors. The aim of this study is to analyse the safety and satisfaction of women included in the PreQ-20 trial who underwent NAC and who underwent mastectomy and immediate reconstruction with prepectoral polyurethane implants. MATERIAL AND METHODS: The patients included in the study belong to the prospective study PreQ-20. The study group consisted of patients who underwent immediate reconstruction after primary systemic therapy. The control groups consisted of patients with immediate reconstruction and adjuvant chemotherapy (control group 1) and patients with an infiltrating carcinoma or in situ ductal carcinoma who did not require chemotherapy (control group 2). RESULTS: The study included 157 women, 58 (36.9%) of whom underwent primary systemic therapy. The indication for genetic study was significantly greater for the study group (87.9%) than for control groups 1 (49.1%) or 2 (30.4%). Seventy-two (45.9%) of the patients underwent bilateral mastectomy (BM), a procedure that was performed significantly more frequently in the study group (69%) than in control groups 1 (30.2%) or 2 (34.8%). The incidence rate for BM after complete pathologic response was 78%. There were no statistically significant differences in the number of complications between the groups. Implant loss was significantly more frequent in control group 1 (13.2%) than in the study group (3.4%) and control group 2 (2.2%). CONCLUSIONS: Mastectomy with prepectoral polyurethane implant reconstruction in patients with neoadjuvant chemotherapy presented a similar incidence of complications compared with patients who underwent primary surgery. There is a high rate of BM in women with NAC.

PSC-derived intestinal organoids with apical-out orientation as a tool to study nutrient uptake, drug absorption and metabolism.

Intestinal organoids recapitulate many features of the in vivo gastrointestinal tract and have revolutionized in vitro studies of intestinal function and disease. However, the restricted accessibility of the apical surface of the organoids facing the central lumen (apical-in) limits studies related to nutrient uptake and drug absorption and metabolism. Here, we demonstrate that pluripotent stem cell (PSC)-derived intestinal organoids with reversed epithelial polarity (apical-out) can successfully recapitulate tissue-specific functions. In particular, these apical-out organoids show strong epithelial barrier formation with all the major junctional complexes, nutrient transport and active lipid metabolism. Furthermore, the organoids express drug-metabolizing enzymes and relevant apical and basolateral transporters. The scalable and robust generation of functional, apical-out intestinal organoids lays the foundation for a completely new range of organoid-based high-throughput/high-content in vitro applications in the fields of nutrition, metabolism and drug discovery.

Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis.

The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders (APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB-/- and MTTP-/- organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets.

Recaídas locorregionales tras mastectomías preservadoras y reconstrucción inmediata en mujeres con cáncer de mama / Locoregional relapse after sparing mastectomies and immediate reconstruction in women with breast cancer

Introducción: Durante los últimos años, los cambios culturales de la sociedad actual y la mejora en la valoración del riesgo han incrementado la indicación de las mastectomías en mujeres con cáncer de mama. Diversos estudios han confirmado la seguridad oncológica de la mastectomías preservadoras y reconstrucción inmediata. El objetivo de este estudio es analizar la incidencia de recaídas locorregionales de este procedimiento y su impacto en la reconstrucción y la supervivencia global. Pacientes y métodos: Estudio prospectivo de pacientes con un carcinoma de mama que realizaron una mastectomía preservadora y reconstrucción inmediata. Se analizaron las recaídas locorregionales, el tratamiento de las mismas y la capacidad de preservar la reconstrucción, así como su impacto en la supervivencia. Resultados: El grupo a estudio lo constituyen 271 mujeres con carcinoma mamario tratadas mediante una mastectomía ahorradora de piel y reconstrucción inmediata. El seguimiento medio fue de 7,98 años y durante el mismo se diagnosticaron 18 recaídas locorregionales (6,6%): 72,2% en el colgajo de la mastectomía y 27,8% ganglionares. No se evidenciaron diferencias significativas en las características patológicas del tumor primario entre las pacientes con y sin una recaída locorregional, aunque el porcentaje de mujeres con tumores hormonosensibles fue superior en el grupo sin recaída. Las pacientes con recaída ganglionar presentaban tumores de mayor tamaño (el 80% T2-T3) y el 60% tenían metástasis axilares al diagnóstico vs. 7,7% de las mujeres con recaída en piel (p = 0,047). Todas las pacientes intervenidas de una recaída locorregional preservaron su reconstrucción. La incidencia de metástasis y muertes fue significativamente mayor en las pacientes con una recaída, causando una disminución no significativa de la supervivencia global. (AU)

Introduction: In recent years, cultural changes in today's society and improved risk assessment have increased the indication for mastectomies in women with breast cancer. Various studies have confirmed the oncological safety of sparing mastectomies and immediate reconstruction. The objective of this study is to analyze the incidence of locoregional relapses of this procedure and its impact on reconstruction and overall survival. Patients and methods: Prospective study of patients with breast carcinoma who underwent a sparing mastectomy and immediate reconstruction. Locoregional relapses and their treatment and their impact on survival were analyzed. Results: The study group is made up of 271 women with breast carcinoma treated with a skin-sparing mastectomy and immediate reconstruction. The mean follow-up was 7.98 years and during the same 18 locoregional relapses (6.6%) were diagnosed: 72.2% in the mastectomy flap and 27.8% lymph node. There were no significant differences in the pathological characteristics of the primary tumor between patients with and without locoregional relapse, although the percentage of women with hormone-sensitive tumors was higher in the group without relapse. Patients with lymph node relapse had larger tumors (80% T2–T3) and 60% had axillary metastases at diagnosis, compared to 7.7% of women with skin relapse (p = 0.047). All patients operated on for locoregional relapse preserved their reconstruction. The incidence of metastases and deaths was significantly higher in patients with a relapse, causing a non-significant decrease in overall survival. (AU)

Extracellular histone release by renal cells after warm and cold ischemic kidney injury: Studies in an ex-vivo porcine kidney perfusion model.

Extracellular histones are cytotoxic molecules involved in experimental acute kidney injury. In patients receiving a renal transplant from donors after circulatory death, who suffer from additional warm ischemia, worse graft outcome is associated with higher machine perfusate extracellular histone H3 concentrations. We now investigated temperature-dependent extracellular histone release in an ex vivo porcine renal perfusion model, and subsequently studied histone release in the absence and presence of non-anticoagulant heparin. Seven pairs of ischemically damaged porcine kidneys were machine perfused at 4°C (cold ischemia) or 28°C (warm ischemia). Perfusate histone H3 concentration was higher after warm as compared to cold ischemia (median (IQR) = 0.48 (0.20-0.83) µg/mL vs. 0.02 (0.00-0.06) µg/mL; p = .045, respectively). Employing immune-electron microscopy (EM), histone containing cytoplasmic protrusions of tubular and endothelial cells were found after warm ischemic injury. Furthermore, abundant histone localization was detected in debris surrounding severely damaged glomerular cells, in a "buck shot" pattern. In vitro, histones were cytotoxic to endothelial and kidney epithelial cells in a temperature-dependent manner. In a separate ex vivo experiment, addition of heparin did not change the total histone H3 levels observed in the perfusate but revealed a continuous increase in the level of a lower molecular weight histone H3 variant. Our findings show that ischemically damaged kidneys release more extracellular histones in warm ischemia, which by EM was due to histone release by renal cells. Blocking of histone-mediated damage during transplantation may be beneficial in prevention of renal injury.

Antenatal Ureaplasma infection induces ovine small intestinal goblet cell defects: a strong link with NEC pathology.

Disruption of the intestinal mucus barrier and intestinal epithelial endoplasmic reticulum (ER) stress contribute to necrotizing enterocolitis (NEC). Previously, we observed intestinal goblet cell loss and increased intestinal epithelial ER stress following chorioamnionitis. Here, we investigated how chorioamnionitis affects goblet cells by assessing their cellular characteristics. Importantly, goblet cell features are compared with those in clinical NEC biopsies. Mucus thickness was assessed as read-out of goblet cell function. Fetal lambs were intra-amniotically (IA) infected for 7d at 122 gestational age with Ureaplasma parvum serovar-3, the main microorganism clinically associated with chorioamnionitis. After preterm delivery, mucus thickness, goblet cell numbers, gut inflammation, epithelial proliferation and apoptosis and intestinal epithelial ER stress were investigated in the terminal ileum. Next, goblet cell morphological alterations (TEM) were studied and compared to human NEC samples. Ileal mucus thickness and goblet cell numbers were elevated following IA UP exposure. Increased pro-apoptotic ER stress, detected by elevated CHOP-positive cell counts and disrupted organelle morphology of secretory cells in the intestinal epithelium, was observed in IA UP exposed animals. Importantly, comparable cellular morphological alterations were observed in the ileum from NEC patients. In conclusion, UP-driven chorioamnionitis leads to a thickened ileal mucus layer and mucus hypersecretion from goblet cells. Since this was associated with pro-apoptotic ER stress and organelle disruption, mucus barrier alterations seem to occur at the expense of goblet cell resilience and may therefore predispose to detrimental intestinal outcomes. The remarkable overlap of these in utero findings with observations in NEC patients underscores their clinical relevance.