Validation of a functional human AD model with four AD therapeutics utilizing patterned iPSC-derived cortical neurons integrated with microelectrode arrays.

Preclinical methods are needed for screening potential Alzheimer's disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. A potential functional parameter to be monitored is long-term potentiation (LTP), which is a correlate of learning and memory, that would be one of the first functions effected by AD onset. Mature human iPSC-derived cortical neurons and primary astrocytes were co-cultured on microelectrode arrays (MEA) where surface chemistry was utilized to create circuit patterns connecting two adjacent electrodes to model LTP function. LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aß42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aß42 induced LTP impairment. The results presented here illustrate the significance of the system as a validated platform that can be utilized to model and study MCI AD pathology, and potentially for the pre-MCI phase before the occurrence of significant cell death. It also has the potential to become an ideal platform for high content therapeutic screening for other neurodegenerative diseases.

Ultramassive transfusion and adjunctive therapies in a case of blood bank depletion.

Background: We present the case of a patient who presents with a high velocity thoracoabdominal gunshot wound requiring ultramassive transfusion who exhausted the county blood bank requiring adjunctive therapies to balanced blood product transfusion while additional blood products could be obtained. Summary: Thoracoabdominal gunshot wounds carry a high mortality of 14-37 % because of the risk to produce cardiopulmonary, solid organ as well as major vascular injuries (Mandal and Oparah (1989) [1]). Ultramassive transfusion (>20 units of blood product transfusion) also carries high morbidity and mortality and management has generally centered on balanced transfusion (Matthay et al. (2021) [2]). Conclusion: Balanced blood product transfusion reduces mortality for patients requiring ultramassive transfusion but when this is not possible utilization of adjuncts to blood products may temporize resuscitation until additional blood products can be obtained.

Development of a functional human induced pluripotent stem cell-derived nociceptor MEA system as a pain model for analgesic drug testing.

The control of severe or chronic pain has relied heavily on opioids and opioid abuse and addiction have recently become a major global health crisis. Therefore, it is imperative to develop new pain therapeutics which have comparable efficacy for pain suppression but lack of the harmful effects of opioids. Due to the nature of pain, any in vivo experiment is undesired even in animals. Recent developments in stem cell technology has enabled the differentiation of nociceptors from human induced pluripotent stem cells. This study sought to establish an in vitro functional induced pluripotent stem cells-derived nociceptor culture system integrated with microelectrode arrays for nociceptive drug testing. Nociceptors were differentiated from induced pluripotent stem cells utilizing a modified protocol and a medium was designed to ensure prolonged and stable nociceptor culture. These neurons expressed nociceptor markers as characterized by immunocytochemistry and responded to the exogenous toxin capsaicin and the endogenous neural modulator ATP, as demonstrated with patch clamp electrophysiology. These cells were also integrated with microelectrode arrays for analgesic drug testing to demonstrate their utilization in the preclinical drug screening process. The neural activity was induced by ATP to mimic clinically relevant pathological pain and then the analgesics Lidocaine and the opioid DAMGO were tested individually and both induced immediate silencing of the nociceptive activity. This human-based functional nociceptive system provides a valuable platform for investigating pathological pain and for evaluating effective analgesics in the search of opioid substitutes.

Classical Complement Pathway Inhibition in a "Human-On-A-Chip" Model of Autoimmune Demyelinating Neuropathies.

Chronic autoimmune demyelinating neuropathies are a group of rare neuromuscular disorders with complex, poorly characterized etiology. Here we describe a phenotypic, human-on-a-chip (HoaC) electrical conduction model of two rare autoimmune demyelinating neuropathies, chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN), and explore the efficacy of TNT005, a monoclonal antibody inhibitor of the classical complement pathway. Patient sera was shown to contain anti-GM1 IgM and IgG antibodies capable of binding to human primary Schwann cells and induced pluripotent stem cell derived motoneurons. Patient autoantibody binding was sufficient to activate the classical complement pathway resulting in detection of C3b and C5b-9 deposits. A HoaC model, using a microelectrode array with directed axonal outgrowth over the electrodes treated with patient sera, exhibited reductions in motoneuron action potential frequency and conduction velocity. TNT005 rescued the serum-induced complement deposition and functional deficits while treatment with an isotype control antibody had no rescue effect. These data indicate that complement activation by CIDP and MMN patient serum is sufficient to mimic neurophysiological features of each disease and that complement inhibition with TNT005 was sufficient to rescue these pathological effects and provide efficacy data included in an investigational new drug application, demonstrating the model's translational potential.

A functional hiPSC-cortical neuron differentiation and maturation model and its application to neurological disorders.

The maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicular glutamate transporter 1 [VGlut1]) and inhibitory (GABA) markers, while late-stage cultures (day 40) expressed solely VGlut1, indicating a purely excitatory phenotype without containing glial cells. This maturation process was further validated utilizing patch clamp and MEA analysis. Particularly, induced long-term potentiation (LTP) successfully persisted for 1 h in day 40 cultures, but only achieved LTP in the presence of the GABAA receptor antagonist picrotoxin in day 21 cultures. This system was also applied to epilepsy modeling utilizing bicuculline and its correction utilizing the anti-epileptic drug valproic acid.

Using Preoperative Pelvic Floor Assessment to Predict Early Return of Continence after Robotic Radical Prostatectomy.

OBJECTIVE: To evaluate whether pre-operative pelvic floor physical therapy (PFPT) parameters may predict early return of urinary continence after RP. While long-term urinary continence is eventually achieved in most patients who undergo radical prostatectomy (RP), predicting when patients will become continent is challenging. Prior studies aiming to predict return of post-operative continence have not evaluated patient-specific pelvic floor strength parameters. METHODS: We reviewed a prospectively maintained database of patients undergoing RP who underwent pre-operative PFPT consultation and completed 3-month patient-reported quality of life evaluation. Trained therapists documented pelvic strength parameters. Urinary continence was defined as using 0 or 1 pad per day. We evaluated the association of PFPT parameters with urinary continence at 3 months, adjusting for other factors that could affect continence. RESULTS: 144 men met inclusion criteria. The majority of patients underwent nerve-sparing procedures and had intermediate- or high-risk prostate cancer. At 3 months, 90 of 144 (62.5%) were continent, while 54 of 144 (37.5%) were not. On multivariable analysis, prostate volume (OR 0.98, 95% CI 0.96-1.00) and pelvic floor endurance (OR 2.71, 95% CI 1.23-6.17) were significantly associated with being continent at 3 months. 56 of 76 (74%) men with good pelvic floor endurance were continent at 3 months, while only 34 of 68 (50%) men with poor endurance were continent (P = .006). CONCLUSION: Pre-operative assessment of pelvic floor endurance is an objective measure that may allow more accurate prediction of early continence after radical prostatectomy. Improved patient counseling could positively impact patient satisfaction and quality of life and reduce decision regret.

Is It Safe to Perform Lung Surgery During the Coronavirus Pandemic?

BACKGROUND:  Coronavirus disease (COVID-19) patients are rapidly growing in our community. Patients with compromised lungs and older age are supposedly at high risk of poor outcomes with COVID-19. We aimed to evaluate the COVID-19 impact on lung surgery during this pandemic at our hospital. METHODOLOGY: This is a retrospective study of all lung surgery patients at our hospital in Boca Raton over three months (February to April 2020). All patients who remained for at least one-day inpatient post-lung surgery were assessed to see if they had an increased incidence of coronavirus infection during the hospital stay or at the follow-up office visit. RESULTS: A total of 44 patients underwent thoracic surgery. It was found that there was no incidence of coronavirus infection in these patients. CONCLUSION: With adequate precautions, older patients can undergo lung surgery during this pandemic. There was no incidence of COVID-19 found among the patients during the hospital stay or at the first follow-up in the office. Also, the postoperative course was not adversely affected.

A human induced pluripotent stem cell-derived cortical neuron human-on-a chip system to study Aß42 and tau-induced pathophysiological effects on long-term potentiation.

INTRODUCTION: The quest to identify an effective therapeutic strategy for neurodegenerative diseases, such as mild congitive impairment (MCI) and Alzheimer's disease (AD), suffers from the lack of good human-based models. Animals represent the most common models used in basic research and drug discovery studies. However, safe and effective compounds identified in animal studies often translate poorly to humans, yielding unsuccessful clinical trials. METHODS: A functional in vitro assay based on long-term potentiation (LTP) was used to demonstrate that exposure to amyloid beta (Aß42) and tau oligomers, or brain extracts from AD transgenic mice led to prominent changes in human induced pluripotent stem cells (hiPSC)-derived cortical neurons, notably, without cell death. RESULTS: Impaired information processing was demonstrated by treatment of neuron-MEA (microelectrode array) systems with the oligomers and brain extracts by reducing the effects of LTP induction. These data confirm the neurotoxicity of molecules linked to AD pathology and indicate the utility of this human-based system to model aspects of AD in vitro and study LTP deficits without loss of viability; a phenotype that more closely models the preclinical or early stage of AD. DISCUSSION: In this study, by combining multiple relevant and important molecular and technical aspects of neuroscience research, we generated a new, fully human in vitro system to model and study AD at the preclinical stage. This system can serve as a novel drug discovery platform to identify compounds that rescue or alleviate the initial neuronal deficits caused by Aß42 and/or tau oligomers, a main focus of clinical trials.

A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing.

Loss of the neuromuscular junction (NMJ) is an early and critical hallmark in all forms of ALS. The study design was to develop a functional NMJ disease model by integrating motoneurons (MNs) differentiated from multiple ALS-patients' induced pluripotent stem cells (iPSCs) and primary human muscle into a chambered system. NMJ functionality was tested by recording myotube contractions while stimulating MNs by field electrodes and a set of clinically relevant parameters were defined to characterize the NMJ function. Three ALS lines were analyzed, 2 with SOD1 mutations and 1 with a FUS mutation. The ALS-MNs reproduced pathological phenotypes, including increased axonal varicosities, reduced axonal branching and elongation and increased excitability. These MNs formed functional NMJs with wild type muscle, but with significant deficits in NMJ quantity, fidelity and fatigue index. Furthermore, treatment with the Deana protocol was found to correct the NMJ deficits in all the ALS mutant lines tested. Quantitative analysis also revealed the variations inherent in each mutant lines. This functional NMJ system provides a platform for the study of both fALS and sALS and has the capability of being adapted into subtype-specific or patient-specific models for ALS etiological investigation and patient stratification for drug testing.

Piezoelectric BioMEMS Cantilever for Measurement of Muscle Contraction and for Actuation of Mechanosensitive Cells.

A piezoelectric biomedical microelectromechanical system (bioMEMS) cantilever device was designed and fabricated to act as either a sensing element for muscle tissue contraction or as an actuator to apply mechanical force to cells. The sensing ability of the piezoelectric cantilevers was shown by monitoring the electrical signal generated from the piezoelectric aluminum nitride in response to the contraction of iPSC-derived cardiomyocytes cultured on the piezoelectric cantilevers. Actuation was demonstrated by applying electrical pulses to the piezoelectric cantilever and observing bending via an optical detection method. This piezoelectric cantilever device was designed to be incorporated into body-on-a-chip systems.

Investigation of the effect of hepatic metabolism on off-target cardiotoxicity in a multi-organ human-on-a-chip system.

Regulation of cosmetic testing and poor predictivity of preclinical drug studies has spurred efforts to develop new methods for systemic toxicity. Current in vitro assays do not fully represent physiology, often lacking xenobiotic metabolism. Functional human multi-organ systems containing iPSC derived cardiomyocytes and primary hepatocytes were maintained under flow using a low-volume pumpless system in a serum-free medium. The functional readouts for contractile force and electrical conductivity enabled the non-invasive study of cardiac function. The presence of the hepatocytes in the system induced cardiotoxic effects from cyclophosphamide and reduced them for terfenadine due to drug metabolism, as expected from each compound's pharmacology. A computational fluid dynamics simulation enabled the prediction of terfenadine-fexofenadine pharmacokinetics, which was validated by HPLC-MS. This in vitro platform recapitulates primary aspects of the in vivo crosstalk between heart and liver and enables pharmacological studies, involving both organs in a single in vitro platform. The system enables non-invasive readouts of cardiotoxicity of drugs and their metabolites. Hepatotoxicity can also be evaluated by biomarker analysis and change in metabolic function. Integration of metabolic function in toxicology models can improve adverse effects prediction in preclinical studies and this system could also be used for chronic studies as well.

Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics.

There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle's contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs.

A comparison of preliminary oncologic outcome and postoperative complications between patients undergoing either open or robotic radical cystectomy.

PURPOSE: To compare complications and outcomes in patients undergoing either open radical cystectomy (ORC) or robotic-assisted radical cystectomy (RRC). MATERIALS AND METHODS: We retrospectively identified patients that underwent ORC or RRC between 2003- 2013. We statistically compared preliminary oncologic outcomes of patients for each surgical modality. RESULTS: 92 (43.2%) and 121 (56.8%) patients underwent ORC and RRC, respectively. While operative time was shorter for ORC patients (403 vs. 508 min; p<0.001), surgical blood loss and transfusion rates were significantly lower in RRC patients (p<0.001 and 0.006). Length of stay was not different between groups (p=0.221). There was no difference in the proportion of lymph node-positive patients between groups. However, RRC patients had a greater number of lymph nodes removed during surgery (18 vs. 11.5; p<0.001). There was no significant difference in the incidence of pre-existing comorbidities or in the Clavien distribution of complications between groups. ORC and RRC patients were followed for a median of 1.38 (0.55-2.7) and 1.40 (0.58-2.59) years, respectively (p=0.850). During this period, a lower proportion (22.3%) of RRC patients experienced disease recurrence vs. ORC patients (34.8%). However, there was no significant difference in time to recurrence between groups. While ORC was associated with a higher all-cause mortality rate (p=0.049), there was no significant difference in disease-free survival time between groups. CONCLUSIONS: ORC and RRC patients experience postoperative complications of similar rates and severity. However, RRC may offer indirect benefits via reduced surgical blood loss and need for transfusion.

A comparison of preliminary oncologic outcome and postoperative complications between patients undergoing either open or robotic radical cystectomy

ABSTRACT Purpose: To compare complications and outcomes in patients undergoing either open radical cystectomy (ORC) or robotic-assisted radical cystectomy (RRC). Materials and Methods: We retrospectively identified patients that underwent ORC or RRC between 2003- 2013. We statistically compared preliminary oncologic outcomes of patients for each surgical modality. Results: 92 (43.2%) and 121 (56.8%) patients underwent ORC and RRC, respectively. While operative time was shorter for ORC patients (403 vs. 508 min; p<0.001), surgical blood loss and transfusion rates were significantly lower in RRC patients (p<0.001 and 0.006). Length of stay was not different between groups (p=0.221). There was no difference in the proportion of lymph node-positive patients between groups. However, RRC patients had a greater number of lymph nodes removed during surgery (18 vs. 11.5; p<0.001). There was no significant difference in the incidence of pre-existing comorbidities or in the Clavien distribution of complications between groups. ORC and RRC patients were followed for a median of 1.38 (0.55-2.7) and 1.40 (0.582.59) years, respectively (p=0.850). During this period, a lower proportion (22.3%) of RRC patients experienced disease recurrence vs. ORC patients (34.8%). However, there was no significant difference in time to recurrence between groups. While ORC was associated with a higher all-cause mortality rate (p=0.049), there was no significant difference in disease-free survival time between groups. Conclusions: ORC and RRC patients experience postoperative complications of similar rates and severity. However, RRC may offer indirect benefits via reduced surgical blood loss and need for transfusion.

A 10-Item Checklist Improves Reporting of Critical Procedural Elements during Transurethral Resection of Bladder Tumor.

PURPOSE: Previous studies have demonstrated significant variation in recurrence rates after transurethral resection of bladder tumor, likely due to differences in surgical quality. We sought to create a framework to define, measure and improve the quality of transurethral resection of bladder tumor using a surgical checklist. MATERIALS AND METHODS: We formed a multi-institutional group of urologists with expertise with bladder cancer and identified 10 critical items that should be performed during every high quality transurethral bladder tumor resection. We prospectively implemented a 10-item checklist into practice and reviewed the operative reports of such resections performed before and after implementation. Results at all institutions were combined in a meta-analysis to estimate the overall change in the mean number of items documented. RESULTS: The operative notes for 325 transurethral bladder tumor resections during checklist use were compared to those for 428 performed before checklist implementation. Checklist use increased the mean number of items reported from 4.8 to 8.0 per resection, resulting in a mean increase of 3.3 items (95% CI 1.9-4.7) on meta-analysis. With the checklist the percentage of reports that included all 10 items increased from 0.5% to 27% (p <0.0001). Surgeons who reported more checklist items tended to have a slightly higher proportion of biopsies containing muscle, although not at conventional significance (p = 0.062). CONCLUSIONS: The use of a 10-item checklist during transurethral resection of bladder tumor improved the reporting of critical procedural elements. Although there was no clear impact on the inclusion of muscle in the specimen, checklist use may enhance surgeon attention to important aspects of the procedure and be a lever for quality improvement.

Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs.

We report on a functional human model to evaluate multi-organ toxicity in a 4-organ system under continuous flow conditions in a serum-free defined medium utilizing a pumpless platform for 14 days. Computer simulations of the platform established flow rates and resultant shear stress within accepted ranges. Viability of the system was demonstrated for 14 days as well as functional activity of cardiac, muscle, neuronal and liver modules. The pharmacological relevance of the integrated modules were evaluated for their response at 7 days to 5 drugs with known side effects after a 48 hour drug treatment regime. The results of all drug treatments were in general agreement with published toxicity results from human and animal data. The presented phenotypic culture model exhibits a multi-organ toxicity response, representing the next generation of in vitro systems, and constitutes a step towards an in vitro "human-on-a-chip" assay for systemic toxicity screening.

Experienced Open vs Early Robotic-assisted Laparoscopic Radical Prostatectomy: A 10-year Prospective and Retrospective Comparison.

OBJECTIVE: To undertake a prospective/retrospective comparison of longer-term oncologic and quality of life outcomes in open radical prostatectomy (ORP) or robotic-assisted laparoscopic radical prostatectomy (RALP) patients. MATERIALS AND METHODS: The clinical progression of ORP and RALP patients who underwent surgery during 2004 was followed over an extended (10 year) period. Pre- and perioperative parameters, oncologic outcomes, recurrence, mortality, and quality of life were compared between surgical modalities. Follow-up time was calculated from the time of surgery to the latest contact. Postoperative quality of life data were obtained from Expanded Prostate Cancer Index Composite survey questionnaires. Recurrence rates, times to recurrence, surgical time, length of stay, hematocrit, follow-up time, and sexual and urinary bother scores were compared between surgical groups. Multivariate analyses were used to predict positive surgical margins and biochemical recurrence. RESULTS: 63 ORP and 116 RALP patients were included (mean age of 60.4 ± 6.4 and 58.6 ± 5.8 years; P = .067), with follow-up times of 10.3 and 10.1 years (P = .191). RALP patients had longer operative times (P < .001), shorter hospital stays (P < .001), and higher discharge hematocrits (P < .001). With prostate-specific antigen, Gleason score, and T-stage as covariates, time to recurrence (P = .365) and positive margin rate (P = .230) were not statistically different between groups. Ninety-five percent of RALP patients were continent and 48.0% were potent vs 92.6% and 41.5% of ORP patients (P = .720; .497). Urinary and sexual bother were not significantly different between groups (P = .392; .985). CONCLUSION: Our longer-term follow-up data suggest that ORP and RALP patients have comparable oncologic and quality of life outcomes.

Multiple intra-renal artery aneurysms causing renal obstruction managed with radical nephrectomy.

A 46-year-old male with a history of hypertension presented with symptoms of persistent abdominal fullness and a non-pulsatile abdominal mass. Subsequent computed tomographic angiography studies revealed the presence multiple large renal aneurysms from the segmental branches of the renal artery and an enlarged hydronephrotic kidney with minimal parenchyma. The renal deterioration appeared to be as a result of an obstruction caused by the large intra-renal aneurysms at the level of the renal calyces. Since the right kidney had no function, an open radical nephrectomy was subsequently performed without complications at 3 months follow up.