Pregnancy and fetal outcomes following maternal exposure to glatiramer acetate in all three trimesters of pregnancy.

BACKGROUND AND PURPOSE: Data on disease-modifying therapy (DMT) exposure throughout pregnancy in patients with multiple sclerosis are scarce. In this analysis, we assessed pregnancy and fetal outcomes following maternal glatiramer acetate (GA) exposure in all three trimesters among cases reported between 1997 and 2020. METHODS: Pregnancy reports of maternal in utero exposure to 20 and 40 mg/mL GA in all three trimesters from 1997 to 2020 were eligible. Both prospective pregnancy data, reported prior to knowledge of pregnancy outcome, and retrospective data were included. The primary endpoint was major congenital malformations (MCMs) based on the European Surveillance of Congenital Anomalies and Twins (EUROCAT) classification. Additional endpoints included fetal death, preterm birth, and low birth weight. The MCM rate was compared to the EUROCAT background rate. RESULTS: A total of 618 GA-exposed pregnancies in all three trimesters resulted in 634 fetuses, including 14 twin pregnancies. One fetal death was reported. All 414 fetuses with data reported prior to knowledge of pregnancy outcome (prospective data) were live births and no fetal death was reported. Preterm birth was reported in 23/213 (10.8%) pregnancies with known gestational age. Low birth weight was reported in 13/203 (6.4%) infants with known birth weight. The prevalence of MCM in prospective live births ranged from 2.2% to 2.4%, which was similar to background rates (2.1%-3.0%). The frequency of these pregnancy and infant outcomes was comparable across GA doses. CONCLUSIONS: In utero exposure to 20 and 40 mg/mL GA in three trimesters of pregnancy does not appear to be related to adverse pregnancy or infant outcomes.

Improving awareness of kidney function through electronic urine output monitoring: a comparative study.

BACKGROUND: The current classification for acute kidney injury (AKI) according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria integrates both serum creatinine (SCr) and urine output (UO). Most reports on AKI claim to use KDIGO guidelines but fail to include the UO criterion. It has been shown that patients who had intensive UO monitoring, with or without AKI, had significantly less cumulative fluid volume and fluid overload, reduced vasopressor use, and improved 30-day mortality. We examined whether real-time monitoring of this simple, sensitive, and easy-to-use biomarker in the ICU led to more appropriate intervention by healthcare providers and better outcomes.  METHODS: RenalSense Clarity RMS Consoles were installed in the General ICU at the Hadassah Medical Center, Israel, from December 2019 to November 2020. The Clarity RMS system continuously and electronically monitors UO in real-time. 100 patients were randomly selected from this period as the study group (UOelec) and compared to a matched control group (UOmanual) from the same period two years earlier. To test whether there was an association between oliguric hours and fluid treatment in each group, the correlation was calculated and analyzed for each of the different UO monitoring methods. RESULTS: Therapeutic intervention: The correlation of the sum of all oliguric hours on Day 1 and 2 with the sum of any therapeutic intervention (fluid bolus or furosemide) showed a significant correlation for the study group UOelec (P = 0.017). The matched control group UOmanual showed no such correlation (P = 0.932). Length of Stay (LOS): Median LOS [IQR] in the ICU of UOelec versus UOmanual was 69.46 [44.7, 125.9] hours and 116.5 [62.46, 281.3] hours, respectively (P = 0.0002). CONCLUSIONS: The results of our study strongly suggest that ICU patients had more meaningful and better medical intervention, and improved outcomes, with electronic UO monitoring than with manual monitoring.

Methionine aminopeptidase 2 as a potential target in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive metastatic cancer with a very low survival rate. This tumor is hypovascularized and characterized by severe hypoxic regions, yet these regions are not impeded by the oxidative stress in their microenvironment. PDA's high resilience raises the need to find new effective therapeutic targets. This study investigated the suitability of methionine aminopeptidase 2 (MetAp2), a metallopeptidase known to play an important role in tumor progression, as a new target for treating PDA. In our examination of patient-derived PDA tissues, we found that MetAp2 is highly expressed in metastatic regions compared with primary sites. At the cellular level, we found that the basal expression levels of MetAp2 in pancreatic cancer cells were higher than its levels in endothelial cells. Pancreatic cancer cells showed a significant suppression of proliferation in a dose-dependent manner upon exposure to TNP-470, a selective MetAp2 inhibitor. In addition, a significant reduction in glutathione (GSH) levels - known for its importance in alleviating oxidative stress - was detected in all treated cells, suggesting a possible anti-cancer activity mechanism that would be feasible for treating highly hypoxic PDA tumors. Furthermore, in an orthotopic pancreatic cancer murine model, systemic oral treatment with a MetAp2 inhibitor significantly reduced tumors' growth. Taken together, our findings indicate that MetAp2 enhances tumor sensitivity to hypoxia and may provide an effective target for treating hypoxic tumors with high expression levels of MetAp2.

Pregnancy, Fetal, and Infant Outcomes Following Maternal Exposure to Glatiramer Acetate During Pregnancy and Breastfeeding.

INTRODUCTION: Published data support the safety of glatiramer acetate in patients with multiple sclerosis who are pregnant or breastfeeding, but long-term data are limited. OBJECTIVE: We aimed to assess pregnancy, fetal, and infant outcomes following maternal exposure to glatiramer acetate. METHODS: In-utero glatiramer acetate-exposed postmarketing pregnancy reports from 2019 to 2021 were extracted from Teva's pharmacovigilance database. Pregnancy data acquired prior to knowledge of pregnancy outcome or detection of congenital malformation (prospective reports) were used to estimate pregnancy and infant outcome rates for glatiramer acetate 20- and 40-mg/mL exposure. A subgroup of cases completed follow-up questionnaires and were analyzed separately. RESULTS: Prospective cases with 702 fetuses had known outcomes with 647 (92.2%) live births, 47 (6.7%) spontaneous abortions, 4 (0.6%) induced abortions, 2 (0.3%) ectopic pregnancies, and 2 (0.3%) fetal deaths. Rates of major congenital malformation (1.1%), preterm births (7.2%), and low/very low birth weight (4.8%), and parameters of growth were within background rates. No infant developmental delay was reported. Overall, pregnancy and infant outcomes were similar across glatiramer acetate doses. CONCLUSIONS: Maternal exposure to glatiramer acetate does not appear to be related to adverse pregnancy, fetal, or infant outcomes. These data further support the safety of both glatiramer acetate 20-mg/mL and 40-mg/mL treatments during pregnancy and breastfeeding.

A new cell-sized support for 3D cell cultures based on recombinant spider silk fibers.

It is now generally accepted that 2D cultures cannot accurately replicate the rich environment and complex tissue architecture that exists in vivo, and that classically cultured cells tend to lose their original function. Growth of spheroids as opposed to 2D cultures on plastic has now been hailed as an efficient method to produce quantities of high-quality cells for cancer research, drug discovery, neuroscience, and regenerative medicine. We have developed a new recombinant protein that mimics dragline spidersilk and that self-assembles into cell-sized coils. These have high thermal and shelf-life stability and can be readily sterilized and stored for an extended period of time. The fibers are flexible, elastic, and biocompatible and can serve as cell-sized scaffold for the formation of 3D cell spheroids. As a proof of concept, recombinant spidersilk was integrated as a scaffold in spheroids of three cell types: primary rat hepatocytes, human mesenchymal stem cells, and mouse L929 cells. The scaffolds significantly reduced spheroid shrinkage and unlike scaffold-free spheroids, spheroids did not disintegrate over the course of long-term culture. Cells in recombinant spidersilk spheroids showed increased viability, and the cell lines continued to proliferate for longer than control cultures without spidersilk. The spidersilk also supported biological functions. Recombinant spidersilk primary hepatocyte spheroids exhibited 2.7-fold higher levels of adenosine triphosphate (ATP) continued to express and secrete albumin and exhibited significantly higher basal and induced CYP3A activity for at least 6 weeks in culture, while control spheroids without fibers stopped producing albumin after 27 days and CPY3A activity was barely detectable after 44 days. These results indicate that recombinant spidersilk can serve as a useful tool for long-term cell culture of 3D cell spheroids and specifically that primary hepatocytes can remain active in culture for an extended period of time which could be of great use in toxicology testing.

Early identification of acute kidney injury in the ICU with real-time urine output monitoring: a clinical investigation.

BACKGROUND: KDIGO (Kidney Disease: Improving Global Outcomes) provides two sets of criteria to identify and classify acute kidney injury (AKI): serum creatinine (SCr) and urine output (UO). Inconsistencies in the application of KDIGO UO criteria, as well as collecting and classifying UO data, have prevented an accurate assessment of the role this easily available biomarker can play in the early identification of AKI. STUDY GOAL: To assess and compare the performance of the two KDIGO criteria (SCr and UO) for identification of AKI in the intensive care unit (ICU) by comparing the standard SCr criteria to consistent, real-time, consecutive, electronic urine output measurements. METHODS: Ninety five catheterized patients in the General ICU (GICU) of Hadassah Medical Center, Israel, were connected to the RenalSense™ Clarity RMS™ device to automatically monitor UO electronically (UOelec). UOelec and SCr were recorded for 24-48 h and up to 1 week, respectively, after ICU admission. RESULTS: Real-time consecutive UO measurements identified significantly more AKI patients than SCr in the patient population, 57.9% (N = 55) versus 26.4% (N = 25), respectively (P < 0.0001). In 20 patients that had AKI according to both criteria, time to AKI identification was significantly earlier using the UOelec criteria as compared to the SCr criteria (P < 0.0001). Among this population, the median (interquartile range (IQR)) identification time of AKI UOelec was 12.75 (8.75, 26.25) hours from ICU admission versus 39.06 (25.8, 108.64) hours for AKI SCr. CONCLUSION: Application of KDIGO criteria for AKI using continuous electronic monitoring of UO identifies more AKI patients, and identifies them earlier, than using the SCr criteria alone. This can enable the clinician to set protocol goals for earlier intervention for the prevention or treatment of AKI.

Novel 3D embryo implantation model within macroporous alginate scaffolds.

BACKGROUND: Implantation failure remains an unsolved obstacle in reproductive medicine. Previous studies have indicated that estrogen responsiveness, specifically by estrogen receptor alpha (ERα), is crucial for proper implantation. There is an utmost need for a reliable in vitro model that mimics the events in the uterine wall during the implantation process for studying the regulatory mechanisms governing the process. The current two-dimensional and hydrogel-based in vitro models provide only short-term endometrial cell culture with partial functionality. RESULTS: Endometrial biopsies showed an increase in E-cadherin expression on the typical window of implantation of fertile women, compared to negligible expression in recurrent implantation failure (RIF) patients. These clinical results indicated E-cadherin as a marker for receptivity. Three-dimensional (3D) macroporous alginate scaffolds were the base for epithelial endometrial cell-seeding and long-term culture under hormone treatment that mimicked a typical menstrual cycle. The RL95-2 epithelial cell culture in macroporous scaffolds was viable for 3 weeks and showed increased E-cadherin levels in response to estrogen. Human choriocarcinoma (JAR) spheroids were used as embryo models, seeded onto cell constructs and successfully adhered to the RL95-2 cell culture. Moreover, a second model of HEC-1A with low ERα levels, showed lower E-cadherin expression and no JAR attachment. E-cadherin expression and JAR attachment were recovered in HEC-1A cells that were transfected with ERα plasmid. CONCLUSIONS: We present a novel model that enables culturing endometrial cells on a 3D matrix for 3 weeks under hormonal treatment. It confirmed the importance of ERα function and E-cadherin for proper implantation. This platform may serve to elucidate the regulatory mechanisms controlling the implantation process, and for screening and evaluating potential novel therapeutic strategies for RIF.

Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability.

The malignancy potential is correlated with the mechanical deformability of the cancer cells. However, mechanical tests for clinical applications are limited. We present here a Triangular Correlation (TrC) between cell deformability, phagocytic capacity, and cancer aggressiveness, suggesting that phagocytic measurements can be a mechanical surrogate marker of malignancy. The TrC was proved in human prostate cancer cells with different malignancy potential, and in human bladder cancer and melanoma cells that were sorted into subpopulations based solely on their phagocytic capacity. The more phagocytic subpopulations showed elevated aggressiveness ex vivo and in vivo. The uptake potential was preserved, and differences in gene expression and in epigenetic signature were detected. In all cases, enhanced phagocytic and aggressiveness phenotypes were correlated with greater cell deformability and predicted by a computational model. Our multidisciplinary study provides the proof of concept that phagocytic measurements can be applied for cancer diagnostics and precision medicine.

Tissue necrosis and its role in cancer progression.

Great efforts have been made in revealing the mechanisms governing cancer resistance and recurrence. The in-situ effects of cell death, caused by hypoxia and metabolic stress, were largely studied in association with inflammation. However, in this work, we focused on the direct effects of necrosis on cancer promotion and on the tumor microenvironment. The conditions leading to cell necrosis, upon nutrient and oxygen deprivation, were recapitulated in-vitro and were used to generate samples for computational proteomic analysis. Under these conditions, we identified clusters of enriched pathways that may be involved in tumor resistance, leading to cancer recurrence. We show that the content of necrotic cells enhances angiogenesis and proliferation of endothelial cells, induces vasculature, as well as increases migration, invasion, and cell-cell interactions. In-vivo studies, where MDA-MB-231 xenografts were exposed to necrotic lysates, resulted in an increase in both proliferation and angiogenesis. Histological analysis of tumor tissues revealed high expression levels of key mediators that were identified by proteomic analysis. Moreover, when cells were injected systemically, coupled with necrotic lysates, a higher number of large lesions was detected in the lung. Finally, using xenografts, we demonstrated that combining an antagonist of a necrotic signal with an anticancer treatment potentiates the prolonged therapeutic effect. This approach suggests a paradigm shift in which targeting late necrotic-secreted factors may increase survival and enhance the efficacy of anticancer therapy.

Tumor cells and their crosstalk with endothelial cells in 3D spheroids.

Recapitulating the tumor microenvironment is a central challenge in the development of experimental model for cancer. To provide a reliable tool for drug development and for personalized cancer therapy, it is critical to maintain key features that  exist in the original tumor. Along with this effort, 3-dimentional (3D) cellular models are being extensively studied. Spheroids are self-assembled cell aggregates that possess many important components of the physiological spatial growth and cell-cell interactions. In this study we aimed to investigate the interconnection between tumor and endothelial cells (EC) in hybrid spheroids containing either tumor cell (TC) lines or patient derived cancer cells. Preparation protocols of hybrid spheroids were optimized and their morphology and tissue-like features were analyzed. Our finding show that capillary-like structures are formed upon assembly and growth of TC:EC spheroids and that spheroids' shape and surface texture may be an indication of spatial invasiveness of cells in the extra-cellular matrix (ECM). Establishing a model of hybrid tumor/stroma spheroids has a crucial importance in the experimental approach for personalized medicine, and may offer a reliable and low-cost method for the goal of predicting drug effects.

Binary Encoding of Random Peptide Sequences for Selective and Differential Antimicrobial Mechanisms.

Binary encoding of peptide sequences into differential antimicrobial mechanisms is reported. Such sequences are random in composition, but controllable in chain length, are assembled from the same two amino acids, but differ in the stereochemistry of one. Regardless of chirality, the sequences lyse bacteria including the "superbugs" methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Sequences with the same chirality, so-called homochiral sequences, assemble into antimicrobial pores and form contiguous helices that are biologically promiscuous and hemolytic. By contrast, heterochiral sequences that lack such persistence selectively attack bacterial membranes without oligomerizing into visible pores. These results offer a mechanistic rationale for designing membrane-selective and sequence-independent antimicrobials.

Rigidity of polymer micelles affects interactions with tumor cells.

Controlling the interaction of drug delivery systems (DDS) with tissues is critical for the success of therapies. Specifically in cancer, due to the high density of the tumors, tissue penetration of DDS is critical and may be challenging. In previous work we have shown that Solidified Polymer Micelles (SPMs) rapidly internalize into cells and tissues. Using AFM analysis, in the present work we measured differences in rigidity of SPM compared with Wet Polymer Micelles (WPM). We further examined whether the semi-solid form of hydrated SPMs has an effect on the interaction with tumor cells both in mono-layer systems and in multi-layer clusters of cells as spheroids. For that we have performed detailed characterization of SPM compared to WPM, including examinations of particle size, stability, drug release kinetics and cell transcytosis, in melanoma A-375 cells. Cell uptake measurements were done using fluorescent signal analysis, FACS and microscopy imaging, showing enhanced abilities of SPMs to penetrate cells and tissues. A simple physical model is presented that well agrees with the experiments and provides insight about the role of particle rigidity in the engulfment mechanism. We conclude that particle rigidity enhances cellular uptake and tissue penetration and that SPMs have a promising potential as an effective and highly permeable DDS. Our findings can be important in future rational design of DDS for particle adjustment to specific tissues and pathologies.

Random peptide mixtures inhibit and eradicate methicillin-resistant Staphylococcus aureus biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is a biofilm-forming pathogen that can cause serious health complications in humans, ranging from minor to life-threatening infections. The challenge of successfully combating biofilms requires the discovery of compounds with a novel mode of action. We have recently developed sequence-random hydrophobic-cationic peptides that display a broad antibacterial activity. In the current study we show that our novel compounds are capable of controlling and managing MRSA biofilms and might be used as lead biofilm inhibitor candidates for further studies.