Photodynamic therapy （PDT） is a tumor treatment modality in which a tumor- localized photosensitizer is excited with light, which results in local production of reactive oxygen species, destruction ...of tumor vasculature, tumor hypoxia, tumor cell death, and induction of an anti-tumor immune response. However, pre-existing tumor hypoxia may desensitize tumors to PDT by activating the hypoxia-inducible factor 1 （HIF-1） survival pathway. Therefore, we hypothesized that inhibition of HIF-1 with acriflavine （ACF） would exacerbate cell death in human epidermoid carcinoma （A431） cells. PDT of A431 tumor cells was per- formed using newly developed and optimized PEGylated cationic liposomes containing the photosensitizer zinc phthalocyanine （ZnPC）. Molecular docking revealed that ACF binds to the dimerization domain of HIF-la, and confocal microscopy confirmed translocation of ACF from the cytosol to the nucleus under hypoxia. HIF-1 was stabilized in hypoxic, but not normoxic, A431 cells following PDT. Inhibition of HIF-1 with ACF increased the extent of PDT-induced cell death under hypoxic conditions and reduced the expression of the HIF-1 target genes VEGF, PTGS2, and EDN1. Moreover, co-encapsulation of ACF in the aqueous core of ZnPC-containing liposomes yielded an adjuvant effect on PDT efficacy that was comparable to non-encapsulated ACF. In conclusion, HIF-1 contributes to A431 tumor cell survival following PDT with liposomal ZnPC. Inhibition of HIF-1 with free or liposomal ACF improves PDT efficacy.
Abstract
Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), by hydrolyzing ...the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), upon receptor stimulation. In plants, PLC’s role is still unclear, especially because the primary targets of both second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, and because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes nine PLCs. We used a reversed-genetic approach to explore PLC’s function in Arabidopsis, and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10–20%. Mutant seeds germinated more slowly but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter–β-glucuronidase (GUS) analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most probably phloem companion cells, in roots, leaves and flowers. In vivo 32Pi labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plant’s tolerance to drought. Our results provide genetic evidence for PLC’s involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed.
Cold sores are nasolabial blisters caused by herpes simplex virus (HSV) infections. Novel therapies demonstrating simultaneously antiviral activity and improved wound healing are warranted. The aim ...of this study was to investigate the efficacy of medical-grade honey (MGH) for treating HSV-induced cold sores. A crossover trial was performed in patients with recurrent cold sores (
= 29). The majority (65.6%) of these patients experience four or more episodes per year, thus forming a valid self-control group. In this study, patients applied an MGH-based formulation (L-Mesitran Soft) on their cold sore at the onset of symptoms (62.1%) or appearing of blister (37.9%) and compared it to their conventional treatments. After complete healing, patients filled in a questionnaire evaluating healing, pain, and itching. The average absolute healing time was 72.4% slower with conventional treatment (10.0 days) compared to MGH (5.8 days). After MGH treatment, 86.2% of all patients experienced faster objective healing (6.9% similar and 6.9% slower) and the subjective healing score was higher in 79.3% of the patients (20.7% similar). If the patients normally experience pain and itching during their cold sores, these levels were lower with MGH therapy compared to conventional treatment in 72.7% and 71.4% of the patients, respectively. Moreover, 100% of the patients prefer MGH treatment over conventional treatment and will use it again on future cold sores. MGH is a promising alternative treatment for cold sores, likely by combining both increased antiviral and wound healing activities while alleviating pain and itching.
Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs ...(uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface) and incision site (control) and blood were obtained from women with uncomplicated pregnancies undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB; genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the concept of site-specific transcriptional adaptation of Tregs within 1 organ.
Little is known about the practice of ventilation management in patients with COVID-19. We aimed to describe the practice of ventilation management and to establish outcomes in invasively ventilated ...patients with COVID-19 in a single country during the first month of the outbreak.
PRoVENT-COVID is a national, multicentre, retrospective observational study done at 18 intensive care units (ICUs) in the Netherlands. Consecutive patients aged at least 18 years were eligible for participation if they had received invasive ventilation for COVID-19 at a participating ICU during the first month of the national outbreak in the Netherlands. The primary outcome was a combination of ventilator variables and parameters over the first 4 calendar days of ventilation: tidal volume, positive end-expiratory pressure (PEEP), respiratory system compliance, and driving pressure. Secondary outcomes included the use of adjunctive treatments for refractory hypoxaemia and ICU complications. Patient-centred outcomes were ventilator-free days at day 28, duration of ventilation, duration of ICU and hospital stay, and mortality. PRoVENT-COVID is registered at ClinicalTrials.gov (NCT04346342).
Between March 1 and April 1, 2020, 553 patients were included in the study. Median tidal volume was 6·3 mL/kg predicted bodyweight (IQR 5·7-7·1), PEEP was 14·0 cm H
O (IQR 11·0-15·0), and driving pressure was 14·0 cm H
O (11·2-16·0). Median respiratory system compliance was 31·9 mL/cm H
O (26·0-39·9). Of the adjunctive treatments for refractory hypoxaemia, prone positioning was most often used in the first 4 days of ventilation (283 53% of 530 patients). The median number of ventilator-free days at day 28 was 0 (IQR 0-15); 186 (35%) of 530 patients had died by day 28. Predictors of 28-day mortality were gender, age, tidal volume, respiratory system compliance, arterial pH, and heart rate on the first day of invasive ventilation.
In patients with COVID-19 who were invasively ventilated during the first month of the outbreak in the Netherlands, lung-protective ventilation with low tidal volume and low driving pressure was broadly applied and prone positioning was often used. The applied PEEP varied widely, despite an invariably low respiratory system compliance. The findings of this national study provide a basis for new hypotheses and sample size calculations for future trials of invasive ventilation for COVID-19. These data could also help in the interpretation of findings from other studies of ventilation practice and outcomes in invasively ventilated patients with COVID-19.
Amsterdam University Medical Centers, location Academic Medical Center.
Aims
Despite clear guideline recommendations for initiating four drug classes in all patients with heart failure (HF) with reduced ejection fraction (HFrEF) and the availability of rapid titration ...schemes, information on real‐world implementation lags behind. Closely following the 2021 ESC HF guidelines and 2023 focused update, the TITRATE‐HF study started to prospectively investigate the use, sequencing, and titration of guideline‐directed medical therapy (GDMT) in HF patients, including the identification of implementation barriers.
Methods and results
TITRATE‐HF is an ongoing long‐term HF registry conducted in the Netherlands. Overall, 4288 patients from 48 hospitals were included. Among these patients, 1732 presented with de novo, 2240 with chronic, and 316 with worsening HF. The median age was 71 years (interquartile range IQR 63–78), 29% were female, and median ejection fraction was 35% (IQR 25–40). In total, 44% of chronic and worsening HFrEF patients were prescribed quadruple therapy. However, only 1% of HFrEF patients achieved target dose for all drug classes. In addition, quadruple therapy was more often prescribed to patients treated in a dedicated HF outpatient clinic as compared to a general cardiology outpatient clinic. In each GDMT drug class, 19% to 36% of non‐use in HFrEF patients was related to side‐effects, intolerances, or contraindications. In the de novo HF cohort, 49% of patients already used one or more GDMT drug classes for other indications than HF.
Conclusion
This first analysis of the TITRATE‐HF study reports relatively high use of GDMT in a contemporary HF cohort, while still showing room for improvement regarding quadruple therapy. Importantly, the use and dose of GDMT were suboptimal, with the reasons often remaining unclear. This underscores the urgency for further optimization of GDMT and implementation strategies within HF management.
Guideline‐directed medical therapy (GDMT) use and dosing in all chronic and worsening heart failure with reduced ejection fraction patients. (A) Percentage of patients using GDMT, stratified by drug class. (B) Reasons for GDMT non‐use. (C) Percentage of target dose for each drug class, stratified by <50% of target dose, 50% to 100% of target dose, and ≥100% of target dose. ARNI, angiotensin receptor–neprilysin inhibitor; BB, beta‐blocker; MRA, mineralocorticoid receptor antagonist; RASi, renin–angiotensin system inhibitor; SGLT2i, sodium–glucose cotransporter 2 inhibitor; TD, target dose.
Abstract Aims Despite clear guideline recommendations for initiating four drug classes in all patients with heart failure (HF) with reduced ejection fraction (HFrEF) and the availability of rapid ...titration schemes, information on real‐world implementation lags behind. Closely following the 2021 ESC HF guidelines and 2023 focused update, the TITRATE‐HF study started to prospectively investigate the use, sequencing, and titration of guideline‐directed medical therapy (GDMT) in HF patients, including the identification of implementation barriers. Methods and results TITRATE‐HF is an ongoing long‐term HF registry conducted in the Netherlands. Overall, 4288 patients from 48 hospitals were included. Among these patients, 1732 presented with de novo, 2240 with chronic, and 316 with worsening HF. The median age was 71 years (interquartile range IQR 63–78), 29% were female, and median ejection fraction was 35% (IQR 25–40). In total, 44% of chronic and worsening HFrEF patients were prescribed quadruple therapy. However, only 1% of HFrEF patients achieved target dose for all drug classes. In addition, quadruple therapy was more often prescribed to patients treated in a dedicated HF outpatient clinic as compared to a general cardiology outpatient clinic. In each GDMT drug class, 19% to 36% of non‐use in HFrEF patients was related to side‐effects, intolerances, or contraindications. In the de novo HF cohort, 49% of patients already used one or more GDMT drug classes for other indications than HF. Conclusion This first analysis of the TITRATE‐HF study reports relatively high use of GDMT in a contemporary HF cohort, while still showing room for improvement regarding quadruple therapy. Importantly, the use and dose of GDMT were suboptimal, with the reasons often remaining unclear. This underscores the urgency for further optimization of GDMT and implementation strategies within HF management.
Objectives
Dental diseases are a major problem in cats and often necessitate tooth extraction. Medical-grade honey (MGH) has antimicrobial and wound-healing properties, and therefore the aim of this ...study was to investigate whether intra-socket application improved healing after tooth extraction. It was postulated that applying MGH would reduce inflammation, improve the viability of the surgical flap and enhance healing following tooth extraction.
Methods
A prospective randomised controlled trial was performed in client-owned cats undergoing bilateral tooth extractions of the same element of the canine or (pre)molar tooth. A split-mouth design was used in which every animal served as its own control. After surgical extraction of the elements, the sockets on one side were filled with an MGH-based ointment (L-Mesitran Soft), whereas the contralateral side received no treatment (control). A mucoperiosteal flap was used on both sides, and simple interrupted monofilament sutures were placed. No antimicrobial drugs were administered. Clinical parameters (inflammation/redness, flap viability and wound healing) were subjectively analysed on days 3 and 7 post-extraction by a veterinarian blinded to the treatment.
Results
Twenty-one cats were included. MGH significantly decreased signs of inflammation (P <0.01), improved mucoperiosteal flap viability (P <0.01) and promoted wound healing (P = 0.01), at both time points. MGH was easy to apply and there were no adverse events.
Conclusions and relevance
Intra-socket application of MGH after tooth extraction positively affects the surgical wound, as it reduces redness, improves flap viability and enhances wound healing. Applying MGH represents a potent adjuvant therapy to support intra-oral wound healing after tooth extraction.
The uterine myometrium is a unique immune environment, capable of harboring a ‘foreign’ fetus without eliciting an immune response.
In this pilot study we aimed to investigate the local presence, ...adaptation and function of human myometrial CD4 + T cells at the maternal-fetal interface in uncomplicated pregnancy.
Myometrial biopsies were obtained at caesarean section from placental and incision site. Lymphocytes were isolated through dissection and digestion with collagenase IV. CD4 + T cells were analyzed by flow cytometry or FACS sorted for RNA sequencing with CEL-seq protocol. Suppression assays were performed with FACS sorted uterine Tregs added to 15,000 healthy donor PBMC in different ratios, in 0.1 μg/mL anti-CD3 coated plates.
Flow cytometry revealed that 70–80% of myometrial CD4 + T cells were CD69 + memory cells, suggesting a tissue-resident memory (TRM) phenotype. RNA sequencing confirmed a TRM-like profile in CD69 + cells, with high expression of CD49a, CXCR6, DUSP6, PD-1 and low expression of CD62L, KLF2/3 and S1PR1 compared to blood memory CD4 + T cells. Especially at the placental site, we observed a high expression of negative costimulatory molecules PD-1, TIGIT, Lag3, TIM-3 and CTLA4 and a high percentage of Tregs. Expression was intermediate at the incision site and low in blood. Suppression assays confirmed the suppressive capacity of myometrial Tregs.
Myometrial CD4 + T cells at the maternal-fetal interface are TRM cells with a high expression of negative costimulatory molecules and a high abundance of functional Tregs. The distribution pattern of these immunoregulatory features indicates that T cells may not only adapt to a tissue, but also to specific sites within one tissue, possibly depending on the local micro-environment.
Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard ...to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.
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