Human Pancreatic Acinar Cells: Proteomic Characterization, Physiologic Responses, and Organellar Disorders in ex Vivo Pancreatitis.

Knowledge of the molecular mechanisms of acute pancreatitis is largely based on studies using rodents. To assess similar mechanisms in humans, we performed ex vivo pancreatitis studies in human acini isolated from cadaveric pancreata from organ donors. Because data on these human acinar preparations are sparse, we assessed their functional integrity and cellular and organellar morphology using light, fluorescence, and electron microscopy; and their proteome by liquid chromatography-tandem mass spectrometry. Acinar cell responses to the muscarinic agonist carbachol (CCh) and the bile acid taurolithocholic acid 3-sulfate were also analyzed. Proteomic analysis of acini from donors of diverse ethnicity showed similar profiles of digestive enzymes and proteins involved in translation, secretion, and endolysosomal function. Human acini preferentially expressed the muscarinic acetylcholine receptor M3 and maintained physiological responses to CCh for at least 20 hours. As in rodent acini, human acini exposed to toxic concentrations of CCh and taurolithocholic acid 3-sulfate responded with trypsinogen activation, decreased cell viability, organelle damage manifest by mitochondrial depolarization, disordered autophagy, and pathological endoplasmic reticulum stress. Human acini also secreted inflammatory mediators elevated in acute pancreatitis patients, including IL-6, tumor necrosis factor-α, IL-1ß, chemokine (C-C motif) ligands 2 and 3, macrophage inhibitory factor, and chemokines mediating neutrophil and monocyte infiltration. In conclusion, human cadaveric pancreatic acini maintain physiological functions and have similar pathological responses and organellar disorders with pancreatitis-causing treatments as observed in rodent acini.

Effect of pessary use on hydronephrosis in women with advanced pelvic organ prolapse: a self-selected interventional trial.

INTRODUCTION AND HYPOTHESIS: The purpose of this study was to determine the proportion of women who demonstrate improvement in hydronephrosis after pessary placement for advanced pelvic organ prolapse (POP). METHODS: This was a planned subset analysis of a prospective study on the prevalence of hydronephrosis in women with advanced POP. Women with anterior or apical POP ≥1 cm past the hymenal remnant were enrolled and screened for hydronephrosis. All were offered expectant management, pessary placement or surgery. Participants self-selecting pessary placement were compared with those with expectant management during the study period. A follow-up ultrasound scan was performed after >3 weeks of treatment. The proportions of participants demonstrating cure/improvement were compared using Fisher's exact test. RESULTS: Of 180 participants enrolled, 55 had hydronephrosis for a prevalence of 30.6% (95% CI 24.3-37.6%). Of those with hydronephrosis, 39 (70.8%) chose pessary placement while 16 (30.2%) declined. A follow-up ultrasound scan was performed in 89% of participants at a median of 77 days (interquartile range 49-99 days). Intention-to-treat analysis showed no difference in improvement or resolution of hydronephrosis between women who accepted and those who declined pessary placement (p = 0.43). However, of 22 women successfully using a pessary, 77.3% showed improvement or cure, compared with 29.6% of 27 women not using a pessary (p = 0.003). CONCLUSIONS: Successful pessary use improved hydronephrosis in over 75% of women with advanced POP. Approximately 39% of women with prolapse did not comply with pessary use and did not demonstrate hydronephrosis improvement.

Severe arsenic poisoning due to Ayurvedic supplements.

Patients that are taking Ayurvedic supplements have an increased risk of heavy metal toxicity. Lead, arsenic, and mercury are frequently identified in these supplements and can cause clinically significant toxicity. Clinicians should screen patients routinely for use of non-pharmaceutical medications and supplements.

Discovery of pyrazolopyrrolidinones as potent, broad-spectrum inhibitors of Leishmania infection.

Introduction: Leishmaniasis is a parasitic disease that affects more than 1 million people worldwide annually, predominantly in resource-limited settings. The challenge in compound development is to exhibit potent activity against the intracellular stage of the parasite (the stage present in the mammalian host) without harming the infected host cells. We have identified a compound series (pyrazolopyrrolidinones) active against the intracellular parasites of Leishmania donovani and L. major; the causative agents of visceral and cutaneous leishmaniasis in the Old World, respectively. Methods: In this study, we performed medicinal chemistry on a newly discovered antileishmanial chemotype, with over 100 analogs tested. Studies included assessments of antileishmanial potency, toxicity towards host cells, and in vitro ADME screening of key drug properties. Results and discussion: Members of the series showed high potency against the deadliest form, visceral leishmaniasis (approximate EC50 ≥ 0.01 µM without harming the host macrophage up to 10.0 µM). In comparison, the most efficient monotherapy treatment for visceral leishmaniasis is amphotericin B, which presents similar activity in the same assay (EC50 = 0.2 µM) while being cytotoxic to the host cell at 5.0 µM. Continued development of this compound series with the Discovery Partnership with Academia (DPAc) program at the GlaxoSmithKline Diseases of the Developing World (GSK DDW) laboratories found that the compounds passed all of GSK's criteria to be defined as a potential lead drug series for leishmaniasis. Conclusion: Here, we describe preliminary structure-activity relationships for antileishmanial pyrazolopyrrolidinones, and our progress towards the identification of candidates for future in vivo assays in models of visceral and cutaneous leishmaniasis.

Identification of Leucinostatins from Ophiocordyceps sp. as Antiparasitic Agents against Trypanosoma cruzi.

Safe and effective treatments for Chagas disease, a potentially fatal parasitic infection associated with cardiac and gastrointestinal pathology and caused by the kinetoplastid parasite Trypanosoma cruzi, have yet to be developed. Benznidazole and nifurtimox, which are currently the only available drugs against T. cruzi, are associated with severe adverse effects and questionable efficacy in the late stage of the disease. Natural products have proven to be a rich source of new chemotypes for other infectious agents. We utilized a microscopy-based high-throughput phenotypic screen to identify inhibitors of T. cruzi from a library of natural product samples obtained from fungi procured through a Citizen Science Soil Collection Program (https://whatsinyourbackyard.org/) and the Great Lakes (USA) benthic environment. We identified five leucinostatins (A, B, F, NPDG C, and NPDG D) as potent inhibitors of the intracellular amastigote form of T. cruzi. Leucinostatin B also showed in vivo suppression of T. cruzi in a mouse model of Chagas disease. Given prior reports that leucinostatins A and B have antiparasitic activity against the related kinetoplastid Trypanosoma brucei, our findings suggest a potential cross-trypanocidal compound class and provide a platform for the further chemical derivatization of a potent chemical scaffold against T. cruzi.

Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies.

Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development.

Dysregulation of Glycerophosphocholines in the Cutaneous Lesion Caused by Leishmania major in Experimental Murine Models.

Cutaneous leishmaniasis (CL) is the most common disease form caused by a Leishmania parasite infection and considered a neglected tropical disease (NTD), affecting 700,000 to 1.2 million new cases per year in the world. Leishmania major is one of several different species of the Leishmania genus that can cause CL. Current CL treatments are limited by adverse effects and rising resistance. Studying disease metabolism at the site of infection can provide knowledge of new targets for host-targeted drug development. In this study, tissue samples were collected from mice infected in the ear or footpad with L. major and analyzed by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differences in overall metabolite profiles were noted in the ear at the site of the lesion. Interestingly, lesion-adjacent, macroscopically healthy sites also showed alterations in specific metabolites, including selected glycerophosphocholines (PCs). Host-derived PCs in the lower m/z range (m/z 200-799) showed an increase with infection in the ear at the lesion site, while those in the higher m/z range (m/z 800-899) were decreased with infection at the lesion site. Overall, our results expanded our understanding of the mechanisms of CL pathogenesis through host metabolism and may lead to new curative measures against infection with Leishmania.

Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Detection of carbohydrates and steroids by cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for biofluid analysis and tissue imaging.

Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO(3) on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desorption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na(+) cationization of carbohydrates and Ag(+) cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1-200 microM). Additionally, we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ion-coated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.

Long term follow-up of Trypanosoma cruzi infection and Chagas disease manifestations in mice treated with benznidazole or posaconazole.

Chagas' Disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for up to 41% of the heart failures in endemic areas in South America and is an emerging infection in regions of North America, Europe, and Asia. Treatment is suboptimal due to two factors. First, the lack of an adequate biomarker to predict disease severity and response to therapy; and second, up to 120-days treatment course coupled with a significant incidence of adverse effects from the drug currently used. Because the disease can manifest itself clinically a few years to decades after infection, controversy remains concerning the suitability of current drug treatment (benznidazole), and the efficacy of alternative drugs (e.g. posaconazole). We therefore followed the clinical course, and PCR detection of parasite burden, in a mouse model of infection for a full year following treatment with benznidazole or posaconazole. Efficacy of the two drugs depended on whether the treatment was performed during the acute model or the chronic model of infection. Posaconazole was clearly superior in treatment of acute disease whereas only benznidazole had efficacy in the chronic model. These results have important implications for the design and analysis of human clinical trials, and the use of specific drugs in specific clinical settings.

Molecular dissection of Chagas induced cardiomyopathy reveals central disease associated and druggable signaling pathways.

Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.

Description and development of a nurse-led cardiac assessment team.

A problem was identified where patient care was affected because of delays in receiving specialist cardiology input. This report describes the experience of developing a specialist cardiac assessment where senior cardiac nurses were trained to provide a 24-hour presence in the emergency department (ED). We describe the service and our evaluation of the service. These dedicated specialised nurses can optimise patient management including admission or safely discharge patients with relevant follow-up when necessary. The team also runs three clinics per week with consultant support. The team of 10 nurses provides a cardiology opinion to approximately 400 patients a month in the ED and 100 patients a month in the acute medical unit (AMU). Eighty-seven per cent of patients are seen in the ED within 30 minutes of referral. Approximately 40% of patients reviewed are accepted directly into cardiology beds thus avoiding admission to the AMU. It has been estimated that 6 bed-days are saved each day, which translated to an estimated £400,000 each year. The team also provides outpatient rapid access services which generates £121,792 income for the directorate. We demonstrate that a cardiac nurse assessment team can provide a cost-effective 24-hour presence in the ED.

Scaffold and Parasite Hopping: Discovery of New Protozoal Proliferation Inhibitors.

Utilizing a target repurposing and parasite-hopping approach, we tested a previously reported library of compounds that were active against Trypanosoma brucei, plus 31 new compounds, against a variety of protozoan parasites including Trypanosoma cruzi, Leishmania major, Leishmania donovani, and Plasmodium falciparum. This led to the discovery of several compounds with submicromolar activities and improved physicochemical properties that are early leads toward the development of chemotherapeutic agents against kinetoplastid diseases and malaria.

Efficient neutralization of antibody-resistant forms of anthrax toxin by a soluble receptor decoy inhibitor.

A soluble receptor decoy inhibitor (RDI), comprised of the extracellular I domain of ANTXR2, is a candidate anthrax therapeutic. Here we show that RDI can effectively neutralize altered forms of the protective antigen toxin subunit that are resistant to 14B7 monoclonal antibody neutralization. These data highlight the potential of RDI to act as an adjunct to existing antibody-based therapies and indicate that inhibitors based on RDI might be useful as a stand-alone treatment against specifically engineered strains of Bacillus anthracis.

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine.

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.

Perception of Caring Among Patients and Nurses.

BACKGROUND: Empirical evidence supports the contention that implementing caring nurse behaviors results in improved patient experience; however, previous studies find differences between patient and nurse perceptions of caring. SIGNIFICANCE: Good patient experience is positively related to desired clinical and financial outcomes. Nurse caring is a critical component in the patient experience. OBJECTIVE: The purposes of this project were to evaluate the congruency between nurse and patient perceptions of nurse caring in a long-term acute care hospital and to determine how much patient perception of nurse caring changes over time. METHOD: The study employed mixed methods using a triangulation strategy in which quantitative data from patients and qualitative data from nurses were collected simultaneously and compared for interpretation. RESULTS: Time affected patient perception of caring significantly. Patients and nurses disagreed about the extent to which nurses ask patients what they know about their illnesses, help them deal with bad feelings, and make them feel comfortable. CONCLUSION: Patients and nurses do not always agree about the quality of caring behaviors, but exposure to nurses over time positively affects patient perception of nurse caring.

The Effect of Lidocaine Gel on Pain Perception During Diagnostic Flexible Cystoscopy in Women: A Randomized Control Trial.

OBJECTIVE: The objective of this study was to determine if there is a clinically meaningful variation in pain perception when using lidocaine gel versus plain lubricant prior to office-based diagnostic flexible cystoscopy. METHODS: This was a randomized, controlled, double-blind trial comparing lidocaine gel and water-based lubricant for the performance of diagnostic flexible cystoscopy. Women undergoing cystoscopy were randomized to either transurethral 2% lidocaine (Uro-Jet) or water-based lubricant prior to cystoscopy. Participants and physicians were blinded, and pain was assessed using an 11-point numeric rating scale (NRS). A priori sample size calculation indicated the need for 40 patients per group to achieve 90% power. Descriptive statistics and Student t test were utilized. RESULTS: The study included 116 patients, 61 in the lidocaine group and 55 in the plain lubricant group. Numeric rating scale in the lidocaine group (2.43 [SD, 1.95]) was significantly lower than that in the plain lubricant group (3.58 [SD, 2.73]) (P = 0.01). After controlling for age and ethnicity, separately as well as together, the procedural NRS scores were 1.37 points (P = 0.002), 0.97 points (P = 0.04), and 1.22 points (P = 0.01) lower in those receiving lidocaine. After the procedure, fewer patients in the lidocaine group (2/61 = 3.3%) requested pain medicine when compared with the plain lubricant group (11/55 = 20%) (P = 0.01). Although anticipated pain scored similarly between groups, actual pain compared with anticipated pain was significantly lower in the lidocaine group (P = 0.02). CONCLUSIONS: Pain perception during flexible cystoscopy decreased when using 2% transurethral lidocaine gel. The use of 2% lidocaine gel is suggested for the reduction of pain at the time of diagnostic flexible cystoscopy in women.

Anthrax toxin receptor 2-dependent lethal toxin killing in vivo.

Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis.

Denervation of skin in neuropathies: the sequence of axonal and Schwann cell changes in skin biopsies.

We compared the pathological changes in cutaneous axons and Schwann cells of individuals with nerve transection to the changes in patients with chronic neuropathies. Following axotomy there was segmentation of axons in the epidermis and dermis on the first day, and loss of axons from the skin was virtually complete by Day 11. Epidermal and small superficial dermal axons were lost before larger caliber and deeper dermal axons. Within the first 50 days following nerve transection, the denervated Schwann cells in the dermis were easily identified by their markers p75 and S100, but by 8 months they had largely disappeared. The chronic neuropathy patients had distally predominant fibre loss, with greater loss of epidermal and dermal fibres in the distal regions of the leg than proximal regions. Several patients had large axonal swellings, often alternating with axonal attenuation, even in regions with normal or nearly normal fibre densities. By electron microscopy the swellings contained accumulations of mitochondria and other particulate organelles as well as neurofilaments. These swellings are likely to represent predegenerative changes in sites of impaired axonal transport, and previous data indicate that the swellings presage fibre loss in the subsequent months. Some of the severely denervated regions had remaining Schwann cells, as judged by immunocytochemistry and by electron microscopy, but others lacked Schwann cells. By analogy with animal experiments, these regions are likely to have had more prolonged denervation. The distribution of axonal loss, the axonal swellings and the changes in Schwann cells all have implications for the design of clinical trials of agents intended to protect cutaneous innervation and to promote regeneration of cutaneous axons in peripheral neuropathies.

4-aminopyridyl-based lead compounds targeting CYP51 prevent spontaneous parasite relapse in a chronic model and improve cardiac pathology in an acute model of Trypanosoma cruzi infection.

BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice. METHODOLOGY/PRINCIPAL FINDINGS: Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart. CONCLUSIONS/SIGNIFICANCE: The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life.