Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing.

BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108-3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

Sirenomelia and maternal chlamydia trachomatis infection: a case report and review.

Background: Sirenomelia is a lethal congenital anomaly, presenting with fusion of lower extremities and malformed perineum. The pathogenesis is unclear, and "defective blastogenesis" is the proposed mechanism. Chlamydia trachomatis (CT) is an obligate intracellular pathogen which reportedly invades placenta and may result in fetal demise. It has documented cytopathogenic effects, specifically, cellular disruption, tissue dysgenesis, and genomic instability.Case report: An infant with sirenomelia was born as a product of 30 weeks of pregnancy, which was normal except for a persistent maternal CT infection. The infant expired shortly after birth.Conclusion: Fetal invasion by CT, conceivably, may induce structural anomalies, such as sirenomelia by virtue of its cytopathic effects. We intend to draw attention to such a possibility by reporting this case. This association, however, is speculative and more cases of sirenomelia with CT positive mothers need to be described in order to make definite conclusions about such a relationship.

Widening the path and window of opportunity for FDA approval of non-vitamin K oral anticoagulant specific antidotes and reversal agents.

There remains a need for safe, immediately effective, and easy to administer antidotes for patients taking novel oral anticoagulants (NOACs) in the settings of major bleeding, need for emergency surgery, and accidental overdose. We review considerations for the successful safety and effectiveness evaluation of potential antidotes currently under development. These compounds are in expedited regulatory approval programs aimed at accelerating the preclinical and clinical evaluation and approval processes for treatments of serious conditions. We review the features of these expedited programs as well as the FDA's efforts to broadly advance the efficiency of drug development and increase the number of new compounds brought to market. The critical path initiative and regulatory science initiative have resulted in numerous successful programs to address current challenges such as a paucity of validated biomarkers and surrogate endpoints as well as unreliable animal models of toxicity. The FDA has also advocated for increased use of pharmacokinetic/pharmacodynamic modeling and adaptive trial design. These efforts foster collaboration between academia, industry and the public sector across interdisciplinary sciences and may continue to widen the pathway for NOAC-specific reversal agents and other novel compounds.

Rebound coagulopathy in patients with snakebite presenting with marked initial coagulopathy.

OBJECTIVE: An estimated 70% of patients with pit viper snakebites require antivenom to treat serious complications such as coagulopathy. Evidence-based guidance is limited for the appropriate administration of Crotalinae Polyvalent Immune Fab (FabAV) and the duration of laboratory follow-up. The objective of our study was to assess the incidence of marked and recurrent envenomation coagulopathy at our trauma center and identify practice patterns that may prevent serious complications. METHODS: A retrospective case review was conducted over a 3-year period on patients treated for symptomatic snakebite injury. Case records were reviewed for the inclusion criteria of international normalized ratio (INR) greater than 2.0. The exclusion criterion was limited to patients receiving anticoagulant therapy. RESULTS: In all, 61 patients were identified on retrospective chart review and 3 patients (4.9%) met inclusion criteria. Two of the 3 patients had marked rebound coagulopathy requiring readmission and additional treatment. In our small series, 2 patients presenting after crotaline envenomation with increased INR (>6.0), decreased fibrinogen (<60 mg/dL), and decreased platelet count (<100,000/mL) had recurrent coagulopathy and were asymptomatic, and recurrence was noted only with follow-up laboratory testing. All patients responded positively within a matter of hours to repeat FabAV administration, with resolution of rebound coagulopathy. CONCLUSIONS: We recommend periodic monitoring of patients with increased INR, decreased fibrinogen, and decreased platelet count. Patients should be monitored for 10 to 14 days after envenomation to identify asymptomatic rebound coagulopathy. Prompt readministration of FabAV appears to correct the coagulopathy.

SpyGlass/Cholangioscope-Assisted Colonoscopic Removal of Appendicolith as a Nonsurgical Alternative for Stump Appendicitis Management.

An appendicolith is a calcified mass which can obstruct the appendix often leading to recurrent appendicitis. Failure to extract fecalith at the time of appendectomy can lead to recurrent inflammation in the appendiceal stump. We describe a 28-year-old man with stump appendicitis due to a retained fecalith postappendectomy. Our approach, akin to interval appendectomy, combines antibiotics and endoscopic intervention postinfection resolution. This case highlights the feasibility and safety of endoscopic management, offering a nonsurgical alternative to prevent the morbidity associated with revision surgery. Further exploration of nonsurgical modalities is warranted to optimize patient outcomes.

Lemmel Syndrome: Unveiling the Underrecognized Pancreatobiliary Diagnosis, Management Strategies, and Future Directions.

Lemmel syndrome, a rare condition, is characterized by biliary obstruction caused by a periampullary diverticulum (a pouch-like outgrowth of the duodenum near the ampulla of Vater). In our case, a 76-year-old male patient presented with epigastric pain and exhibited a cholestatic pattern on liver function tests. Imaging revealed dilated pancreatic and common bile ducts due to compression by a periampullary diverticulum (double duct sign: simultaneous dilation of the common bile duct and pancreatic duct). Upper endoscopy showed one medium-sized periampullary diverticulum. This case emphasizes the diagnostic process and the importance of considering Lemmel syndrome in differential diagnosis in elderly patients with biliary obstruction. We discuss the prevalence, diagnostic considerations, including imaging modalities, and treatment options, emphasizing the need for further research.

Intensive Care Unit-Related Cholangiopathy-Induced Biliary Cast Syndrome Without Liver Transplantation: A Rare Entity.

The development of biliary cast syndrome (BCS) is very rare, mostly documented in patients with liver transplantation. The etiology of BCS is unknown; however, risk factors include post-liver transplant bile duct injury, ischemia, infection, fasting, parenteral feeding, and increased bile viscosity and gallbladder dysmotility. We present the case of a 41-year-old man who developed BCS secondary to a prolonged intensive care unit course without a liver transplant. This case highlights the importance of monitoring patients with protracted intensive care unit course and abnormal aminotransferases to recognize and timely manage cholangiopathy and BCS-related complications.

Decoding the diagnostic dilemma: Peripheral T­cell lymphoma presenting as acute pancreatitis: A case report.

Peripheral T cell lymphoma (PTCL) is a rare form of non-Hodgkin lymphoma characterized by the origin of mature T-cells. PTCL demonstrates atypical clinical features and involves both nodal and extra-nodal sites. The diagnosis and treatment of PTCL can prove to be challenging, as it is often detected at advanced stages and is resistant to conventional chemotherapy treatments. The present report describes a 55-year-old male patient who presented with acute pancreatitis, and imaging suggested a soft tissue mass in the pancreatic head indicating pancreatic adenocarcinoma. Further investigation through ultrasound-guided biopsy led to the diagnosis of pancreatic PTCL not otherwise specified.

Inosine induces stemness features in CAR-T cells and enhances potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

Fabrication and Characterization of Three-Dimensional Macroscopic All-Carbon Scaffolds.

We report a simple method to fabricate macroscopic, 3-D, free standing, all-carbon scaffolds (porous structures) using multiwalled carbon nanotubes (MWCNTs) as the starting materials. The scaffolds prepared by radical initiated thermal crosslinking, and annealing of MWCNTs possess macroscale interconnected pores, robust structural integrity, stability, and conductivity. The porosity of the three-dimensional structure can be controlled by varying the amount of radical initiator, thereby allowing the design of porous scaffolds tailored towards specific potential applications. This method also allows the fabrication of 3-D scaffolds using other carbon nanomaterials such as single-walled carbon nanotubes, fullerenes, and graphene indicating that it could be used as a versatile method for 3-D assembly of carbon nanostructures with pi bond networks.

A Rare Case of 45,X/46,X,del(Y)(q12→qter) Mosaicism in An Infertile Male with Y Chromosome Microdeletion.

Background: Males with 45,X/46,XY karyotype have two different types of cells. This condition is associated with a wide range of clinical phenotypes. In infertile males, the mosaic 45,X/46,XY karyotype is a frequent sex chromosome defect and they might be able to conceive with the help of assisted reproductive technology; nevertheless, there is a potential risk of transmission of azoospermia factor (AZF) microdeletions in addition to 45,X to all the male progeny. In this case report, the purpose was to present a rare sex chromosomal mosaicism of an infertile man. Case Presentation: Comprehensive molecular and cytogenetic analysis of an infertile male was performed in this case study. A 27-year-old male was presented with history of azoospermia and was unable to conceive after being involved in five years of marriage. Cytogenetic investigation revealed a rare mosaic karyotype pattern of 45,X/46,X,del(Y)(q12→qter). Y chromosome microdeletion (YMD) analysis revealed notable deletions of 06 loci. Comparative genomic hybridization (CGH) microarray was performed to investigate probable functional genetic associations. Conclusion: Deletion of Y-linked genes leads to different testicular pathological conditions contributing to male infertility. Individuals with normal male phenotype harbor YMD, although size and location of the deletion do not always correspond well with quality of sperm. Therefore, in addition to semen analysis, identification of genetic variables is important which will play a crucial role in proper diagnosis and management of infertile couples. The present case study demonstrates the significance of comprehensive molecular testing and cytogenetic screening for individuals with idiopathic infertility.

Changes in low-acuity patient volume in an emergency department after launching a walk-in clinic.

Objective: Unscheduled low-acuity care options are on the rise and are often expected to reduce emergency department (ED) visits. We opened an ED-staffed walk-in clinic (WIC) as an alternative care location for low-acuity patients at a time when ED visits exceeded facility capacity and the impending flu season was anticipated to increase visits further, and we assessed whether low-acuity ED patient visits decreased after opening the WIC. Methods: In this retrospective cohort study, we compared patient and clinical visit characteristics of the ED and WIC patients and conducted interrupted time-series analyses to quantify the impact of the WIC on low-acuity ED patient visit volume and the trend. Results: There were 27,211 low-acuity ED visits (22.7% of total ED visits), and 7,058 patients seen in the WIC from February 26, 2018, to November 17, 2019. Low-acuity patient visits in the ED reduced significantly immediately after the WIC opened (P = 0.01). In the subsequent months, however, patient volume trended back to pre-WIC volumes such that there was no significant impact at 6, 9, or 12 months (P = 0.07). Had WIC patients been seen in the main ED, low-acuity volume would have been 27% of the total volume rather than the 22.7% that was observed. Conclusion: The WIC did not result in a sustained reduction in low-acuity patients in the main ED. However, it enabled emergency staff to see low-acuity patients in a lower resource setting during times when ED capacity was limited.

Evaluation of Efficacy of Bromocriptine as a Therapeutic Modality in the Treatment of Diabetes Mellitus: A Systematic Review.

Diabetes mellitus (DM), one of the oldest diseases known to mankind has always been difficult to treat even with the availability of a variety of medications. In such a scenario, the Food and Drug Administration (FDA) has approved a novel therapeutic, bromocriptine, with a different mechanism of action than the traditional medications since 2009 but has not been used as either first-line therapy or add-on therapy. In this systematic review, we searched databases like PubMed, Medline, PubMed Central, Cochrane Library, Clinicaltrials.gov, and Wiley Online Library. The selected articles were screened using inclusion and exclusion criteria and quality appraised; finally, 11 studies including eight clinical trials and three narrative reviews were included. It was found that an increase in dopamine and serotonin levels were hypothesized to convert the insulin-resistant (IR) state to an insulin-sensitive (IS) state. Hence in DM, as there is an IR state, the administration of dopamine was hypothesized to increase insulin sensitivity. In our study based on included studies, it was found that bromocriptine was superior as an add-on therapy to metformin compared to metformin alone, also it was found beneficial in people failing treatment with any one oral hypoglycemic agent. On the contrary, bromocriptine was found inferior to teneligliptin in treating DM. Still, more studies are required to make an accurate and reliable assessment of the efficacy of bromocriptine in treating DM.

Inosine Induces Stemness Features in CAR T cells and Enhances Potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing.

Cardioprotection of electroacupuncture against myocardial ischemia-reperfusion injury by modulation of cardiac norepinephrine release.

Augmentation of cardiac sympathetic tone during myocardial ischemia has been shown to increase myocardial O(2) demand and infarct size as well as induce arrhythmias. We have previously demonstrated that electroacupuncture (EA) inhibits the visceral sympathoexcitatory cardiovascular reflex. The purpose of this study was to determine the effects of EA on left ventricular (LV) function, O(2) demand, infarct size, arrhythmogenesis, and in vivo cardiac norepinephrine (NE) release in a myocardial ischemia-reperfusion model. Anesthetized rabbits (n = 36) underwent 30 min of left anterior descending coronary artery occlusion followed by 90 min of reperfusion. We evaluated myocardial O(2) demand, infarct size, ventricular arrhythmias, and myocardial NE release using microdialysis under the following experimental conditions: 1) untreated, 2) EA at P5-6 acupoints, 3) sham acupuncture, 4) EA with pretreatment with naloxone (a nonselective opioid receptor antagonist), 5) EA with pretreatment with chelerythrine (a nonselective PKC inhibitor), and 6) EA with pretreatment with both naloxone and chelerythrine. Compared with the untreated and sham acupuncture groups, EA resulted in decreased O(2) demand, myocardial NE concentration, and infarct size. Furthermore, the degree of ST segment elevation and severity of LV dysfunction and ventricular arrhythmias were all significantly decreased (P < 0.05). The cardioprotective effects of EA were partially blocked by pretreatment with naloxone or chelerythrine alone and completely blocked by pretreatment with both naloxone and chelerythrine. These results suggest that the cardioprotective effects of EA against myocardial ischemia-reperfusion are mediated through inhibition of the cardiac sympathetic nervous system as well as opioid and PKC-dependent pathways.

Importance of medical interpretation in guiding emergent stroke treatment: a case report.

ABSTRACTEffective communication between patients and providers is critically important, particularly in the setting of acute illness. In time-sensitive medical emergencies, language barriers may be seen as burdensome, but should be addressed attentively to gather accurate information and to form a trusting relationship with patients and their families. Such is the case with a 79-year-old man who was brought to the emergency department (ED) with symptoms concerning for acute stroke. His Russian-speaking wife was the only source of the patient's history, and information was initially gathered without the aid of a medical interpreter. Based on this information, which was provided in broken English, the patient was determined to be out of the time window to receive intravenous tissue plasminogen activator (IV tPA). Subsequent history taken by the admitting medicine team with the help of a medical interpreter revealed that the patient's symptoms began much more recently than previously understood. Based on the time he was last seen normal, the patient could have been a candidate for IV tPA when he first presented, but the information was clarified after he was outside this time window. Subsequent head imaging confirmed the presence of acute infarct in the left middle cerebral artery territory.This communication failure serves to highlight the importance of thorough and detailed communication between healthcare providers and their patients. As evidenced by the case presented, the outcomes of this underutilization can lead to catastrophic decision-making and, in turn, hinder the ability of providers to provide quality care for their patients.

Doomed peripheral intravenous catheters: Bad Outcomes are similar for emergency department and inpatient placed catheters: A retrospective medical record review.

INTRODUCTION: The survivorship of peripheral intravenous catheters (PIVCs) placed in hospitalized patients is shockingly poor and leads to frequent reinsertions. We aimed to evaluate differences in failure rates and IV insertion practices for PIVCs that are placed in the emergency department (ED) compared to those placed in the inpatient (IP) setting. METHODS: We conducted a retrospective electronic medical record review of PIVC survival at a single-site suburban, academic tertiary care referral center with 130,000 annual ED visits and 1100 inpatient beds. Adult patients admitted requiring at least one PIVC were included. The primary outcome was incidence of premature failure of PIVCs. Secondary outcomes included dwell time, completion of therapy, catheter diameter, and site of insertion as they relate to PIVC survival. RESULTS: Between January 2018 and July 2019, 90,743 IV catheters were included from 47,272 unique patient encounters in which 35,798 and 54,945 catheters were placed in the ED and IP units, respectively. There was no significant difference in failure rate between the ED and IP PIVCs, with 53.1% of ED PIVCs failing and 53.4% of IP PIVCs failing (p = 0.35). Mean dwell time for ED PIVCs was 3.4 days compared to a mean of 3.2 days for IP placed PIVCs (p < 0.001). 48% of ED PIVCs achieved completion of therapy at the first insertion compared to 59% of IP PIVCs (p < 0.001). The antecubital fossa and forearm had the lowest failure rate of 53% and 50%, respectively, and 22 gauge PIVCs had the highest failure rate of 60.5%. CONCLUSION: PIVCs have similar poor survival rates regardless of ED versus IP location of the insertion. The forearm and antecubital fossa sites should be preferentially used. Smaller diameter (22G) catheters have highest complications and poorest survival regardless of site of insertion. Larger diameter catheters (18 or 20 gauge) may offer improved outcomes.

Kotter's 8 stages of change: implementation of clinical screening protocols for assessing patients for COVID-19 - a review of an academic medical centre's preparedness.

BACKGROUND: COVID-19 screening protocols rapidly evolved as a result of changing Centers for Disease Control and Prevention (CDC) and California Department of Public Health (CDPH) recommendations. These protocols led to operational improvements at one large academic medical centre using change management methods explained in Kotter's 8-stage change model. METHODS: We reviewed all iterations of clinical process maps for identifying, isolating and assessing COVID-19 infections in paediatric and adult populations within one emergency department (ED) from 28 February 2020 to 5 April 2020. We incorporated CDC and CDPH criteria for the various roles of healthcare workers in ED patient assessment. RESULTS: Using Kotter's 8-stage change model, we outlined the chronological evolution of basic screening criteria, as well as how these were reviewed, modified and implemented during the onset and through the time of greatest uncertainty of COVID-19 in the USA. Our results demonstrate a successful creation, and subsequent execution, of rapidly changing protocols across a large workforce. CONCLUSION: We effectively applied a business change management framework to the hospital management response during a pandemic; we share these experiences and challenges to inform and guide future operational decision making during times of rapid change.