Current Options Research Articles

Fixing the leaky physician-scientist pipeline: Integrated-dedicated research period programs in emergency medicine.

Emergency physicians (EPs) are well positioned to perform medical research. EPs are exposed to a wide range of disease types, medical specialties, and treatment modalities. Furthermore, emergency medicine (EM) serves as the safety net for the U.S. health care system. The diverse exposure provides a vast opportunity for EP to perform many worthwhile research projects. Yet, EM has historically had the lowest amount of funding and a lower number of National Institutes of Health-funded research projects. Many suggest the etiology is a "leaky" educational pipeline with loss of many potential physician-scientists over the training and development course. Current research training options for the EM physician-scientist includes MD-PhD, 4-year EM residency program and postresidency fellowships. While each has its advantages and disadvantages, we describe an additional educational alternative of EM physician-scientists, which we have named the integrated-dedicated research period within an EM residency. We describe the features of these programs and preliminary results from the graduates and current trainees.

RAAV8-PC Gene Therapy for Severe Protein C Deficiency Using a Novel Murine Model

Introduction Protein C (PC) is a vitamin K-dependent protein, synthesized in the liver. The PROC gene is located on chromosome 2 at position 2q13-14 and encodes 9 exons. Severe protein C deficiency (SPCD) is a rare devastating autosomal recessive disease that presents at birth with purpura fulminans (PF) and is associated with high rates of morbidity and mortality. As current management options are limited and burdensome, there is a large unmet need for new treatments including curative therapies. Because small increases in PC may significantly ameliorate the clinical presentation of SPCD and potentially prevent PF, we hypothesize that SPCD could be treated by PC gene therapy (GT). Aim To establish a murine model enabling the survival of SPCD mice and to evaluate PC expression after recombinant adeno-associated viral vector- PC (rAAV8-PC) gene therapy. Methods The study was conducted with C57Bl/6 PC heterozygous mice (a kind gift of Prof. Weiler, [Wisconsin]), severe hemophilia A (HA) mice and wild type C57Bl/6 mice. Genotyping was performed by PCR using appropriate primers for murine PROC and F8 genes. rAAV8-PC was produced by transfecting HEK293 cells with PX-680 as rAAV helper, AAV8 and PC plasmid with an alpha-1 antitrypsin promotor. Extraction and purification were performed using a commercial kit. Viral concentration was determined by a designated commercial qPCR assay. rAAV8 - PC (10 12 /kg Vg of AAV8-PC) was injected via the tail vein to 5-8 weeks old mice. Blood samples were collected from the facial vein into 1.5 ml tubes containing 0.109M sodium citrate at blood: anticoagulant ratio of 1:1 (vol/vol). PC plasma levels were measured prior and following rAAV8-PC infusion using a commercial ELISA assay for PC antigen. Thrombin generation was measured in plasma samples using a modification of the calibrated automated thrombinoscope assay. Results As expected, breeding of heterozygous PC mice yielded only 3/200 homozygous SPCD mice (PC levels= 0), that survived for less than 24 hours. Thus, GT for heterozygous PC mice was considered; however, PC plasma levels in heterozygous PC mice were not substantially different from those in WT mice (median levels in heterozygous PC and WT mice: 216 ng/ml vs. 190 ng/ml, respectively), which precluded this option. Hence, with the aim of increasing the survival rate and duration of homozygous SPCD mice, we decided to rebalance the severe thrombotic disorder of SPCD, by induction of a genetically engineered combination with the severe coagulopathy of Hemophilia. We established an alternative SPCD murine model by breeding severe HA mice with heterozygous PC mice. This approach yielded homozygous SPCD mice with severe HA (PROC-/F8-), exhibiting a significantly prolonged survival compared to homozygous SPCD mice with normal F8 levels (over 90% survived over 6 weeks). Genotyping and ELISA of PC levels confirmed PC homozygosity (null mutation) as well as severe HA. Nineteen PROC-/F8- mice were injected with rAAV8-PC into the tail vein and followed for at least 12 weeks. Nine mice died during blood sampling at different time points and were excluded from further analysis. Individual responses to PC- GT were noted among the remaining 10 mice, reflected by non-uniform PC expression measured by ELISA (initial PC increase, time to peak and PC peak plasma levels). An increase in PC level was noted at week 2-5 post vector infusion, reaching PC peak level between week 2-10 post GT. Table 1 summarizes the individual patterns of 10 PROC-/F8-mice that underwent GT. The functional activity of PC in the murine plasma following GT was supported by thrombin generation assays. Analyses performed in PROC-/F8-mice before and after GT demonstrated endogenous thrombin potential (ETP) and peak hight decrease following GT, as compared to untreated PROC-/F8-mice. Conclusion We present here a novel murine model of combined PC and factor VIII deficiency that supports the survival of SPCD mice and enables PC gene therapy studies. Our results provide the first proof of concept that infusion of rAAV8-PC results in increased PC plasma levels in mice, and may serve as a basis for future studies investigating GT for SPCD. The concept of modulating a severe genetic prothrombotic disorder, which is nearly incompatible with life, by genetic combination with severe coagulopathy may offer a valuable tool enabling investigation and potential development of treatment modalities for the prothrombotic disorder.

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P<0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P<0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P<0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

Current Management of Pregnant Persons with ITP: Systematic Literature Review

Introduction: Immune thrombocytopenia (ITP) often occurs during pregnancy and thrombocytopenia can worsen as the pregnancy progresses. Thrombocytopenia during pregnancy is significant because of concerns regarding risk of postpartum hemorrhage and neonatal thrombocytopenia. Ideally, patients are managed jointly by hematologists and high-risk obstetricians with a treatment goal to minimize the risk of bleeding complications. The 2019 American Society of Hematology ITP guidelines do not address pregnancy. The 2019 International ITP Consensus Report recommends initial therapy with steroids or intravenous immunoglobulin (IVIG), and patients refractory to initial therapy may need anti-D (Rh(D) positive nonsplenectomized persons), combination therapy, thrombopoietin receptor agonists (TPO-RAs), or rituximab in severe cases. Although the 2019 American College of Obstetricians and Gynecologists (ACOG) guidelines agree with initial management, they disagree with 2nd and 3rd line recommendations stating there is not enough evidence to recommend either anti-D or rituximab during pregnancy and do not mention TPO-RAs at all. Therefore, the purpose of this study was to perform a systematic literature review to describe current management of pregnant persons with ITP. Methods: In March 2023, we conducted a systematic search in the MEDLINE, Embase and Web of Science databases to identify articles on treatment of ITP in pregnancy from 2001-present. Keywords included terms for primary ITP and terms for pregnancy. Inclusion criteria included: 1) English language, 2) humans, and 3) at least 10 pregnant persons in the cohort. Exclusion criteria were: 1) did not contain original data, 2) conference abstracts, 3) unable to separate primary vs secondary ITP, and 4) unable to separate data for only the patients managed after 2001. Three persons independently reviewed articles and performed data abstraction. The online Covidence systematic review management system was utilized to standardize data abstraction and determine consensus. Adverse outcomes were abstracted as reported in the original article. Results: After removal of duplicates, a total of 1396 articles were screened for eligibility, 420 full articles were reviewed and 30 met eligibility criteria (Figure 1). Of the included articles, the majority were retrospective cohort studies (18/30), 6 prospective cohort, 2 case-control, 2 cross-sectional, and 2 clinical trials. These 30 articles represented 3282 pregnant persons aged 16-45 years, with 3868 total pregnancies managed across 14 different countries. Fifty-seven percent of pregnancies (2207/3868) required treatment during the pregnancy and of those treated, 36% (805/2207) only required steroids. Other common treatments included: 17% (372/2207) IVIG + steroids, 14% (307/2207) IVIG only, 11% (232/2207) TPO-RAs, and 10% (225/2207) received platelet transfusions. Only 8 pregnancies were treated with rituximab and 0 with anti-D. Nine articles described management during delivery. The majority (58%; 695/1203) reported no treatment was administered during delivery. When treatment was administered during delivery, it was a platelet transfusion (59%; 302/508) or steroids (40%; 204/508). Only 2 maternal deaths were reported. There were 2 neonatal deaths and 68 intrauterine deaths. Also 7% of the total pregnancies were reported to have a post-partum hemorrhage complication. Conclusion: As expected, pregnant persons with ITP were initially managed with steroids and IVIG. However, results from this study suggest hematologists are comfortable treating pregnant persons with off-label TPO-RAs. Patients were rarely treated with rituximab and 0 were treated with anti-D. Pregnancy in ITP was not without maternal risk as 7% of the pregnancies were complicated by hemorrhage. In comparison, the rate of postpartum hemorrhage in 2019 in the United States was reported to be 4.3% (Corbetta-Rastelli, Obstetrics & Gynecology 2023; 141:152-161). Although despite the hemorrhage rate, there were only 2 maternal deaths. Strengths of this study include the number of countries and pregnancies represented; however, limitations include the country-level differences in access to therapies and differences related to risk of pregnancy complications. The treatment landscape for ITP continues to expand; therefore, guidelines reflecting current management options are critical.

Ropeginterferon Alfa-2b Blunts Hyperactive JAK2 Activity in Polycythemia Vera and Essential Thrombocythemia

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPNs) characterized by phenotype defining somatic mutations of JAK2, cMPL, and CALR genes. These mutations activate the JAK-STAT signaling pathway, leading to augmented cellular proliferation. A major cause of morbidity and mortality in PV and ET is thrombosis. Current therapy options include hydroxyurea (HU), interferon-α (IFN-α), and JAK2 inhibitors. IFN-α treatment normalizes blood cell counts, and reduces thrombotic complications in PV and ET and has been associated with a decrease in JAK2V617F variant allele frequency (VAF), indicating its selective targeting of JAK2V617F-mutated cells. IFN-α treatment also rescues the normal dormant HSCs, resulting in conversion of clonal to polyclonal myelopoiesis (Tashi, Leukemia, 2018). Ropeginterferon alfa (ropeg-IFNα)-2b is mono-pegylated IFNα-2b with greater tolerability and an extended half-life compared to Peg-IFNα-2α and is FDA approved for PV therapy. We recently discovered a patient with a novel germline JAK2R715T mutation, which causes erythrocytosis with PV-like features (Kim, ASH, 2022, Song under review in Blood). This patient had poor tolerance to HU and a JAK2 inhibitor therapy; although the latter normalized her blood cell counts, it was poorly tolerated. She has been on ropeg-IFNα-2b since Mar. 2023. Given that ropeg-IFNα-2b's known function is to reduce mutated cells (Verger, Blood Cancer J. 2018) and all her hematopoietic cells were bearing a JAK2 gain-of-function germline mutation, we did not expect a complete hematological response, but her blood counts completely normalized. This observation led us to hypothesize that ropeg-IFNα-2b induces remission by reduction of JAK2 activity. To measure JAK2 activity, we quantitated phosphorylated STAT5 (p-STAT5) levels in her erythroid progenitors (CD71 +/CD235a +) and observed a 20% reduction in these levels after ropeg-IFNα-2b treatment. This prompted us to investigate PV and ET patients with the typical JAK2V617F mutation treated with ropeg-IFNα-2b. We measured p-STAT5 in erythroid progenitors and platelets (CD61 +) of 8 PV and 5 ET patients treated with ropeg-IFNα-2b and 1 PV and 6 ET patients treated with HU. Levels of p-STAT5 were higher in patients treated with HU compared to those treated with Ropeg-IFNα-2b (Figure A and B). In healthy controls, levels of p-STAT5 were lower than those in HU treated patients but comparable to those in ropeg-IFNα-2b treated patients (Figure A and B). p-STAT5 levels showed a positive correlation with platelet counts (r=0.5235, p=0.0397) and neutrophil counts (r=0.6088, p=0.0235). p-STAT5 levels were then measured before and after ropeg-IFNα-2b treatment in 1 ET patient and showed a 20% reduction, which coincided with a decrease in platelet count from 746 x 10 9/L to 207 x 10 9/L. Peg-IFN-2α downregulates upregulated oxidative stress genes and upregulates downregulated antioxidative defense genes in MPNs (Skov, PLOS One, 2022), suggesting its potential role in reducing oxidative stress.We measured reactive oxygen species (ROS) in erythroid progenitors and found that ROS levels were significantly higher in PV and ET patients treated with HU compared to those treated with ropeg-IFNα-2b and to healthy controls (Figure C). JAK2V617F mutated cells accumulate more ROS compared to unmutated cells (Marty, Leukemia, 2013). We found a positive correlation between ROS levels and p-STAT5 levels (r=0.4778, p=0.0449), suggesting an interaction between JAK2 activity and oxidative stress in PV and ET. Our study demonstrated the impact of ropeg-IFNα-2b treatment on JAK2 activity in PV and ET. Ropeg-IFNα-2b effectively reduced JAK2 activity, leading to suppression of dysregulated cell proliferation, and decreased ROS accumulation. To minimize the influence of confounding factors that could independently affect JAK2 activity, we are now comparing samples from the same patients before and after ropeg-IFNα-2b treatment. These results suggest a novel molecular benefit of ropeg-IFNα-2b therapy in PV and ET.

Outcomes of Regimented Weight Monitoring on Morbidity and Mortality during AML Induction

Acute myeloid leukemia (AML) is characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow and other tissues. AML is a disease of elderly, with median age of 67 years at diagnosis. Current therapeutic options for elderly individuals with AML include intensive chemotherapy with a cytarabine and anthracycline backbone, hypomethylating agents (decitabine and azacytidine) alone or in combination with venetoclax. Targeted therapies, low dose cytarabine, investigational agents and supportive care with hydroxyurea and transfusions are other options. While monitoring daily weights has been well documented in APL due to concern for differentiation syndrome, little has been documented in AML. Monitoring daily weights can be a useful tool in maintaining patients' euvolemic status. This can help in appropriately resuscitating patients with AML as they are prone to sepsis due to their immunocompromised status. Also, it can help prevent fluid overload and prevent hypoxic respiratory failure. At our center over the last few years, we have standardized an approach to monitoring and maintaining the weights of our AML patients at or near their pre-admission level with IV fluids and diuresis, as needed. We report outcomes of regimented weight monitoring on morbidity and mortality during AML induction. METHOD We conducted a retrospective chart review of newly diagnosed AML patients treated at our center between January 2016 and May 2022. The cohort was divided into 2 groups; those treated prior to 2019 and those treated after. For each group, patients whose weights were monitored and maintained within 3 kilograms of admission weight were identified for comparison with those who were not. Induction regimens including 7 plus 3 with or without the addition of targeted therapies were reviewed. Patients who were ineligible for induction and those admitted directly to the ICU prior to the start of therapy were excluded from this study. We reviewed the records of 155 patients induced at our center; the mean age at diagnosis was 56 years (20-82). 74 (48%) of these 155 patients were females. Prior to 2019, 73 patients were induced; 68 of these patients did not have monitored weights. Of these 22 were ICU transfers (detailed in Table 2). The 5 remaining patients had weights monitored and none were transferred to the ICU. After January 1st 2019, weights were followed and euvolemia was maintained in 32 patients, of whom 3 (9.4%) were transferred to ICU. Of the 50 patients after 2019 in whom weights were not maintained, 12 were transferred to the ICU (as summarized in Table 1). Out of 155 patients, 118 patients did not have weights followed and had a weight change of more than 3 kilograms above or below from admission. Of these 118 patients, 34 were transferred to ICU. 37 patients had weights followed, of which 3 were transferred to ICU. (OR 0.22) (95% CI 0.06-0.75) (p=0.017). The characteristics, mean length stay (LOS) and reasons for ICU transfer have been detailed in table 1. The mean overall length of stay (LOS) for all patients was 31.21 days. Mean LOS was 23.94(n=37) and 33.50(n=118) days for patients whose weights were and were not followed, respectively (mean difference of 9.56 days) (95 % CI of 4.8115-14.3085) (p<0.01). Nineteen out of 160 patients died due to causes listed in Tables 1 and 2. Among the 37 patients in whom daily weights were maintained, 3 patients, all of whom were transferred to ICU, died during induction. Of 118 patients in whom daily weights were not maintained, 14 patients died in the ICU during induction, whereas 1 patient died in hospice after developing bacteremia and respiratory failure (OR 0.6059, 95 % CI= 0.1653-2.2205, p=0.4496). DISCUSSION Fluid overload is a significant concern in the treatment of AML. Our findings suggest that maintaining euvolemia and monitoring daily weights can effectively manage patients on the floor and prevent unnecessary ICU transfers. Although there are limitations to our study due to its retrospective, single-center nature but the cost-effectiveness and noninvasive mode of maintaining euvolemia support its potential application to standard treatment protocols. As we have demonstrated, this simple intervention may contribute to reducing ICU transfers, LOS and overall mortality during induction therapy for AML patients. Further prospective studies are warranted to validate these findings.

Actionable Findings from an Unbiased Drug Screen for Novel Single Agent and Combination Therapies Against AML with Mecom Re-Arrangement

EVI1 (Ecotropic Viral Integration Site 1) is a zinc finger transcriptional regulator encoded from the MECOM (MDS1 and EVI1 Complex) locus at chromosome 3q26.2. Aberrant EVI1 overexpression is observed in approximately 10% of de novo AML, of which approximately half are due to chromosomal rearrangement involving either inv3(q21;q26.2) or t(3;3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is relocated and drives EVI1 expression. In addition to the resulting haploinsufficiency of GATA2, monosomy 7 and activating mutations in the RAS pathway and SF3B1 commonly co-occur in 3q26.2-rearranged (r) AML. AML with EVI1 overexpression generally have a stem-like phenotype, are refractory to current therapy options and exhibit poor overall survival. Among the targets of EVI1 are the ERG, MYC, KIT, Bcl-xL and MPL genes. GATA2 repression, coupled with increased EVI1 activity, promotes AML progression and an aggressive phenotype in 3q26.2-rearranged AML. Therefore, there is a glaring need to develop and test novel targeted therapies with improved efficacy in repressing EVI1 and improving survival of AML with EVI1 overexpression. To identify druggable vulnerabilities in 3q26.2-r AML, we conducted an unbiased high-throughput drug screen, utilizing a library of 2480 oncology-focused, mechanistically annotated drugs (referred as NCATS Mechanism Interrogation Plates or-MIPE 5.0) (Cancer Cell. 2021; 39:566-579), against seven AML cell lines, i.e., four with 3q26.2-r: UCSD-AML1, HNT-34, AML191 and AML194, and three non-3q26.2-r AML: SET-2, MV4-11 and OCI-AML3. The library exploits mechanistic redundancy by including multiple inhibitors against well-validated oncology targets, while simultaneously encompassing mechanistic diversity, altogether targeting more than 850 distinct mechanisms of action. We exploited this redundancy to identify druggable, target-level dependencies in 3q26.2-r AML, both in terms of absolute potency and relative activity as compared to other AML subtypes. BRD4 was identified as an absolute dependency in 3q26.2-r AMLs. This was consistent with previous reports that BET inhibitors (e.g., OTX015, mivebresib or ABBV-075 and JQ1) are effective against 3q26.2-r AML cell lines, patient-derived (PD) AML cells and PDX models. Comparison of 3q26.2-r versus other AML cell lines also identified XIAP, mTOR, PIK3CA and Bcl-xL as druggable vulnerabilities in 3q26.2-r AML. In follow-up experiments, XIAP/cIAPs inhibitors birinapant (10-1000 nM) or SM-164 (30-1000 nM), chosen based on the MIPE screen outcomes, induced significantly more dose-dependent apoptosis in 3q26.2-r versus the other AML cell lines. This was associated on Western analyses with decline in XIAP, c-IAP1/2, p-ERK1/2, MCL1 and Bcl-xL, but increased protein levels of cleaved caspase-3 and PARP in UCSD-AML1 and AML191 cells. Mivebresib (50-250 nM) or OTX015 (100-500 nM) also induced dose-dependent apoptosis, and reduced EVI1, c-Myc, c-Myb, XIAP, c-IAP1/2, CDK4/6 and Bcl-xL while increasing protein levels of HEXIM1 and cleaved PARP. Consistent with this, co-treatment with birinapant with mivebresib was synergistically lethal in UCSD-AML1 and AML191 cells, with Delta Synergy scores > 1.0 calculated by the ZIP method. Treatment with the dual mTOR/PIK3CA inhibitor NVP-BGT226 (1-30 nM) or navitoclax or Bcl-xL-specific BH3 mimetic A-1155463 also exerted lethality and synergistically induced apoptosis with mivebresib in AML cells with inv3/t(3;3). We previously reported that the TBL1/β-catenin antagonist tegavivint induced apoptosis in 3q26.2-r PD AML cells and PDX models. Co-treatment with birinapant and tegavivint also synergistically induced apoptosis in 3q26.2-r AML cells. Finally, in the xenograft model of AML194-Luc cells in NSG mice, monotherapy with birinapant (30 mg/kg/t.i.w, Intraperitoneal), vs vehicle control, significantly reduced the AML burden, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with birinapant and the BETi OTX015 (30 mg/kg/day, by oral gavage) was more effective in reducing AML burden in the xenograft model. These findings demonstrate promising preclinical activity of IAP protein inhibition against the cellular models of AML with inv3/t(3;3) with EVI1 overexpression, supporting the rationale to further evaluate in vivo efficacy of birinapant and/or BETi-based combinations against this AML sub-type.

Review of the role of biofilms in equine wounds: Clinical indications and treatment strategies

SummaryRecognition of the role that biofilms play in the persistence of wounds and lack of response to therapy in horses is increasing. Prevention of biofilm development in the early stages of wound care involves three primary strategies: wound debridement and cleansing to reduce bacterial counts in the wound bed, appropriate use of advanced wound dressings and implementation of topical antimicrobial agents. Once formed, eradication of biofilms requires elimination to improve the wound environment for contraction and epithelialisation while not further harming the native cells integral to the healing process, which is achieved predominantly through repeated lavage and debridement combined with topical antimicrobial therapy. Pseudomonas, Enterococcus and Staphylococcus bacterial species have been most commonly identified in equine wounds with delayed healing. This review will establish why and how biofilms form, how to recognise clinical indications that biofilms have formed in equine wounds and review current diagnostic options and biofilm‐based wound care strategies to eradicate biofilms. Clinical scenarios for cases in which biofilms developed and were successfully treated will be presented. This review will advance practitioners' understanding of the presence and role of biofilms in equine wounds and provide an updated summary of recommended treatment strategies.

HPB P19 Post-hepatectomy liver failure: A timeline centered review

Abstract Background Post-hepatectomy liver failure (PHLF) is a leading cause of postoperative mortality after liver surgery. Due to its significant impact, it is imperative to understand the risk stratification and preventative strategies for PHLF. Whilst the literature addressing PHLF appears ubiquitous, there is no specific structure that summarizes the evidence that can guide surgeons regarding the timeline of the preventive measures against PHLF. The objective of this review is to summarize the published measures and their sources of evidence, in a timeline centered way around the index (i.e., definitive) hepatectomy, thus, summarizing the practice in prediction, prevention, and treatment. Methods This review includes studies on both humans and animals, where they addressed PHLF. A literature search was conducted across the Cochrane Library, Embase, MEDLINE/PubMed, and Web of Knowledge electronic databases for English language studies published between July 1997 and June 2020. Studies presented in other languages were equally considered. The quality of included publications was assessed using Downs and Black’s checklist. The results were presented in qualitative summaries owing to the lack of studies qualifying for quantitative analysis. Results This systematic review with 245 studies, provides insight into the current prediction, prevention, diagnosis, and management options for PHLF. This review highlighted that liver volume manipulation is the most frequently studied preventive measure against PHLF in clinical practice, with modest improvement in the treatment strategies over the past decade. Conclusions Remnant liver volume manipulation is the most consistent preventive measure against PHLF.

An Overview of Dental Solid Waste Management and Associated Environmental Impacts: A Materials Perspective

Dental healthcare plays an important role in the overall health of individuals, and the sector is rapidly growing around the world due to increases in population, healthcare facilities, and improved access for economically weaker sections of society. Dental procedures and oral care generate a significant amount of biomedical waste that should be managed in an environmentally safe and sustainable manner. An overview is presented of the current status of dental solid waste management with a focus on waste composition from traditional and emerging dental treatments, new-generation dental materials, waste treatment procedures, and current options. Dental waste can be broadly divided into three categories: infectious waste, non-infectious waste, and domestic-type waste. Infectious waste contains materials contaminated with blood or other infectious mouth fluids, amalgam, and sharps, whereas non-infectious dental waste is devoid of human fluid contamination but can be potentially toxic due to the presence of amalgams, acids, metal dust, resins, etc. Suspended particulates in dental wastewater are another likely source of contamination. Appropriate segregation of this waste is essential for containing infections during waste processing. New-generation dental materials, such as nanomaterials, resin-based composites, and ceramics, are finding increasing applications in a variety of dental procedures as antimicrobial, restorative, and therapeutic agents. While incineration and landfilling have been used for processing traditional dental waste, the presence of novel materials in dental waste raises several additional concerns. Novel single/multistage recycling approaches need to be developed for dental waste towards resource recovery, thus minimizing incineration and landfilling to the extent possible.

Glutamine addiction and therapeutic strategies in pancreatic cancer

Pancreatic cancer remains one of the most lethal diseases worldwide owing to its late diagnosis, early metastasis, and poor prognosis. Because current therapeutic options are limited, there is an urgent need to investigate novel targeted treatment strategies. Pancreatic cancer faces significant metabolic challenges, principally hypoxia and nutrient deprivation, due to specific microenvironmental constraints, including an extensive desmoplastic stromal reaction. Pancreatic cancer cells have been shown to rewire their metabolism and energy production networks to support rapid survival and proliferation. Increased glucose uptake and glycolytic pathway activity during this process have been extensively described. However, growing evidence suggests that pancreatic cancer cells are glutamine addicted. As a nitrogen source, glutamine directly (or indirectly via glutamate conversion) contributes to many anabolic processes in pancreatic cancer, including amino acids, nucleobases, and hexosamine biosynthesis. It also plays an important role in redox homeostasis, and when converted to α-ketoglutarate, glutamine serves as an energy and anaplerotic carbon source, replenishing the tricarboxylic acid cycle intermediates. The present study aims to provide a comprehensive overview of glutamine metabolic reprogramming in pancreatic cancer, focusing on potential therapeutic approaches targeting glutamine metabolism in pancreatic cancer.

VEXAS syndrome: Clinical, hematologic features and a practical approach to diagnosis and management.

VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a newly identified disease caused by somatic alterations in UBA1 which produce a recalcitrant inflammatory state along with hematologic disturbances. Patients with VEXAS can have a wide spectrum of clinical symptoms and providers should be familiar with the heterogeneity of associated clinical features. While hematologic parameters may be generally non-specific, peripheral blood features of macrocytosis, monocytopenia, and/or thrombocytopenia coupled with bone marrow vacuolization of erythroid or myeloid precursors should raise suspicion for this condition. Due to an increased mortality, prompt recognition and accurate diagnosis is paramount. Access to testing for confirmation of UBA1 variants is not yet universally available but clinicians should understand the current available options for genetic confirmation of this disease. Treatment options are limited due to lack of prospective clinical trials but cytokine directed therapies such as interleukin-6 inhibitors and JAK-STAT inhibitors as well as hypomethylating agents such as azacitidine have shown evidence of partial effect. Though cases are limited, allogeneic stem cell transplantation holds promise for durable response and potential cure. The intent of this review is to outline the pathophysiology of VEXAS syndrome and to provide a practical approach to diagnosis and treatment.

Emerging Treatments for Childhood Interstitial Lung Disease.

Childhood interstitial lung disease (chILD) is a large and heterogeneous group of disorders characterized by diffuse lung parenchymal markings on chest imaging and clinical signs such as dyspnea and hypoxemia from functional impairment. While some children already present in the neonatal period with interstitial lung disease (ILD), others develop ILD during their childhood and adolescence. A timely and accurate diagnosis is essential to gauge treatment and improve prognosis. Supportive care can reduce symptoms and positively influence patients' quality of life; however, there is no cure for many of the chILDs. Current therapeutic options include anti-inflammatory or immunosuppressive drugs. Due to the rarity of the conditions and paucity of research in this field, most treatments are empirical and based on case series, and less than a handful of small, randomized trials have been conducted thus far. A trial on hydroxychloroquine yielded good safety but a much smaller effect size than anticipated. A trial in fibrotic disease with the multitargeted tyrosine kinase inhibitor nintedanib showed similar pharmacokinetics and safety as in adults. The unmet need for the treatment of chILDs remains high. This article summarizes current treatments and explores potential therapeutic options for patients suffering from chILD.

EXTH-47. TUMOUR-TARGETED PAN-RAS INHIBITION AS A NOVEL THERAPY FOR DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract INTRODUCTION Diffuse Intrinsic Pontine Glioma (DIPG) has a median survival of 9 months following diagnosis in children 7-9 years old. Current therapeutic options are limited to radiation and thus tumour specific vulnerabilities are the subject of ongoing research. One such vulnerability is RAS signalling due to its upregulation in DIPG. RAS regulates cellular proliferation and survival making it an attractive target. Based on diphtheria toxin (DT), we have developed a molecular chimera which selectively binds a DIPG cell surface receptor and releases a RAS cleaving peptidase (RRSP). We hypothesize that using a DIPG targeted RRSP conjugated chimera will reduce tumour viability and improve survival in DIPG models. METHODS Viability of SU-DIPG XIII, XVII, XXV, 36 and 50 cell lines treated with RRSP-DTB, and a DIPG targeted RRSP conjugated chimera was measured using CellTiterGlo Luminescent Assay. Proteomic analysis and single-cell RNA sequencing was used to identify a cell surface receptor upregulated on tumour cell populations from patient samples. SU-DIPG 36 orthotopic xenograft mice were treated with our DIPG targeted RRSP conjugated chimera or vehicle for one week delivered into the pons via osmotic pump and monitored using bioluminescent imaging. RESULTS RRSP-DTB successfully reduces DIPG cell viability in vitro with an EC50 between 0-3 picomolar, demonstrating successful toxicity mediated by RRSP. This is supported by ablation of RAS verified on Western Blot. ANTXR1 receptor was identified as upregulated in tumour cell populations, and a TEM8 conjugated RRSP chimera was developed. RRSP-TEM8 has an EC50 of 5-70 picomolar in vitro and significantly improves survival in vivo with a median survival of 68 days (n = 14) compared to the vehicle control (n = 14) 57 days (p = 0.02). Our results present a successful novel therapeutic as our RRSP chimera, RRSP-TEM8, effectively targets DIPG cells and reduces cell viability to improve survival.

Potential of Natural products for Chemoprevention of Breast Cancer

Background: Breast cancer is the most frequent female cancer and one of the leading causes of cancer death in women. There are many chemotherapy agents available for the treatment of breast cancer. Still, the current therapeutic options have not fulfilled the desired outcomes, especially for drug-resistant breast cancer therapy. Thus, there is an urgent need to develop novel anti-breast cancer agents. Objective: This overview targets to portray the new advancement of herbal and artificial derivatives with anti-breast cancer potential, protecting the articles allotted from the ultimate year. Method: We found significant articles of interest in the field of breast cancer and proposed a review based on relevant information. Description of the studies included in the review. Result: Various key components are found in vegetables, fruits, seeds, nuts, coffee, tea, and wine. Therefore, dietary exposure is significant. Natural products having low-cost toxicity, their availability in foods, and their existence in a variety of herbal treatments, caution should be exercised in further assessment of their properties and applications. Conclusion: We tried to analyze the anti-breast cancer potency of quite a few extracts from different plant sources and to compare their antiproliferative efficiency of crude extracts with the action of their purified ingredients while an additional study is required.

Health care satisfaction and medical literacy habits among caregivers of individuals with Down syndrome.

Patients with Down syndrome have significant specialized health care needs. Our objective was to understand the needs, satisfaction, and online habits of caregivers as they care for persons with Down syndrome. A mixed-method survey was distributed through REDCap from April 2022 to June 2022 in the United States; a Spanish-translated version was distributed through SurveyMonkey from August 2022 to March 2023 in Mexico. We received 290 completed responses from the United States and 58 from caregivers in Mexico. We found that current health care options are not meeting the needs of many individuals with DS in both the United States (39.7%) and Mexico (46.6%). Caregivers expressed frustrations with the inaccessibility and inapplicability of health care information. In particular, they often found the volume of information overwhelming, given their limited medical background. Additionally, health care recommendations were not customized and lacked practical recommendations. Most caregivers in both the United States (72.1%) and Mexico (82.8%) believe it is not easy to find answers to medical questions about their loved ones with DS. Online platforms with customized, specific health information related to DS could offer innovative solutions to these unmet needs for families and primary care providers.

Right heart remodeling and outcomes of patients with tricuspid regurgitation

Abstract Background Albeit long neglected, significant (at least moderate, ≥ 2+) tricuspid regurgitation (TR) is currently known to be associated with increased mortality, and has thus become a relevant therapeutic target [1-3]. Recent and somewhat preliminary evidence has shown, however, that different subtypes of TR might respond differently to current therapeutic options [4-5]. In fact, secondary TR, rather than being a single entity, can develop because of either right ventricular (RV) remodelling (i.e functional TR) or tricuspid annulus (TA) dilation secondary to right atrial (RA) dilation (i.e isolated TR). Purpose The aim of this meta-analysis was to study the association between right heart remodeling and long-term all-cause mortality in a population of patients with significant TR, independent of the treatment option. Methods MEDLINE, ISI Web of Science, and SCOPUS databases were searched for studies published up to July 2022. Studies reporting data on at least one echocardiographic RV anatomic and/or functional parameter and outcome in patients with significant TR were included. This study was designed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) requirements. The primary endpoint was long-term all-cause mortality. Results Out of 3152 studies, a total of 14 were included in the final analytic synthesis, enrolling 4394 subjects. The median follow-up was 33 [24–51] months. Overall, long-term all-cause mortality was 31% (95% confidence interval [CI]: 20-41%, p = <0.001; Figure 1); however, a significant difference was noted in the medical subgroup vs. the surgery subgroup (43% [32-53%] vs 11% [20-41%]). By means of a meta-regression analysis, a significant and inverse relation was found between tricuspid annular plane systolic excursion (TAPSE), RV fractional area change (RV-FAC) and mortality (11 studies enrolling 3551 subjects, coefficient -0.063 [-0.111 to -0.014], p = 0.011; 9 studies, 2975 subjects, coefficient -0.044 [-0.059 to -0.029], p < 0.001, respectively; Figure 2A and 2B), whereas a significant and concordant relation was found between TA dimension, RA area and mortality (7 studies, 2986 subjects, coefficient 0.003 [0.002 to 0.005], p < 0.001; 6 studies, 1920 subjects, coefficient -0.019 [-0.025 to -0.013], p < 0.001, respectively; Figure 2C and 2D). Conclusions The higher mortality associated with right ventricular dysfunction and reduced mortality with increasing right atrial dilatation suggest a better prognostic outcome for isolated TR than functional TR. Careful assessment of right heart anatomy and function is warranted, as it might foster the identification of different subtypes of TR with unique prognoses.Forest plot all-cause mortalityResult of the meta-regression analysis

Inflammation-induced TRIM21 represses hepatic steatosis by promoting the ubiquitination of lipogenic regulators.

Nonalcoholic steatohepatitis (NASH) is a leading cause for chronic liver diseases. Current therapeutic options are limited due to an incomplete mechanistic understanding of how steatosis transitions to NASH. Here we show that the TRIM21 E3 ubiquitin ligase is induced by the synergistic actions of proinflammatory TNF-α and fatty acids in livers of humans and mice with NASH. TRIM21 ubiquitinates and degrades ChREBP, SREBP1, ACC1, and FASN, key regulators of de novo lipogenesis, and A1CF, an alternative splicing regulator of the high-activity ketohexokinase-C (KHK-C) isoform and rate-limiting enzyme of fructose metabolism. TRIM21-mediated degradation of these lipogenic activators improved steatosis and hyperglycemia as well as fructose and glucose tolerance. Our study identifies TRIM21 as a negative regulator of liver steatosis in NASH and provides mechanistic insights into an immunometabolic crosstalk that limits fatty acid synthesis and fructose metabolism during metabolic stress. Thus, enhancing this natural counteracting force of steatosis through inhibition of key lipogenic activators via TRIM21-mediated ubiquitination may provide a therapeutic opportunity to treat NASH.

Inflammation and Psoriasis: A Comprehensive Review.

Psoriasis is an immune-mediated disease with a strong genetic component that brings many challenges to sick individuals, such as chronic illness, and which has multiple associated comorbidities like cardiovascular disease, metabolic syndrome, inflammatory bowel disease, and psychological disorders. Understanding the interplay between the innate and adaptative immune system has led to the discovery of specific cytokine circuits (Tumor Necrosis Factor-alpha (TNF-α), IL-23, IL-17), which has allowed scientists to discover new biomarkers that can be used as predictors of treatment response and pave the way for personalized treatments. In this review, we describe the footprint psoriasis leaves on the skin and beyond, key pathophysiological mechanisms, current available therapeutic options, and drawbacks faced by existing therapies, and we anticipate potential future perspectives that may improve the quality of life of affected individuals.

DIRECT POSTERIOR KNEE PORTALS USING NEEDLE ARTHROSCOPY: A TECHNICAL FEASIBILITY STUDY

Knee arthroscopy is typically approached from the anterior, posteromedial and posterolateral portals. Access to the posterior compartments through these portals can cause iatrogenic cartilage damage and create difficulties in viewing the structures of the posterior compartments. The purpose of this study was to assess the feasibility of needle arthroscopy using direct posterior portals as both working and visualising portals.For workability, the needle scope was inserted advanced from anterior between the cruciate ligament bundle and the lateral wall of the medial femoral condyle until the posterior compartments were visualised. For visualisation, direct postero-lateral and -medial portals were established. The technique was performed in 9 knees by two experienced researchers.Workability and instrumentation of the posteromedial compartment and meniscus was achieved in 56%. The posterior horns could not be visualised in four specimens as the straight lens could not provide a more medial field of view. Visualisation from the direct medial posterior portal allowed a clear view of the medial meniscus, femoral condyle and posterior cruciate ligament in all specimens. Workability and instrumentation of the posterolateral compartment was not possible with the needle scope.Direct posterior approaches for the posteromedial compartment access are challenging with the current needle scope options and could only be achieved in over 50%. The postero-lateral compartment was not accessible. An angled lens or a flexible Needle scope would be better suited for developing this technique further.