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.

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.

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.

Prevalence of Hydronephrosis in Women With Advanced Pelvic Organ Prolapse.

OBJECTIVE: To describe the prevalence of hydronephrosis in advanced pelvic organ prolapse (POP) and to describe clinical and urodynamic parameters associated with hydronephrosis. MATERIALS AND METHODS: Prospective, observational cohort study examining the prevalence of hydronephrosis in advanced POP. Women with a POP-Q examination of at least +1 for points C, Aa, or Ba were enrolled and screened for hydronephrosis. Basic demographics, clinical, and urodynamic findings among women with and without hydronephrosis were compared. The University of Southern California IRB approved this protocol. RESULTS: A total of 180 participants were enrolled. Fifty-five women had some hydronephrosis, for a prevalence of 30.6% (24.3%-37.6%). Mean age was 57.9 (±9.0) years and mean body mass index was 29.2 kg/m(2) (± 4.6). Of the participants, 80.6% were postmenopausal. The presence of diabetes mellitus was significantly associated with hydronephrosis (8% without vs 21.8% with, P = .009), as was the degree of anterior and apical (median Aa, Ba, C, and D higher with hydronephrosis than without, P <.01) but not posterior POP (median Ap and Bp, P = .13, and .2, respectively). On multichannel urodynamics, participants with hydronephrosis had higher mean post void residuals (64.8 vs 38.5 mL, P = .007), lower mean first leak (199.6 vs 280.8 mL, P = .006), and higher mean maximum cystometric capacity (525.2 vs 476.7 mL, P = .02) compared with participants without hydronephrosis. CONCLUSION: The prevalence of hydronephrosis in women with advanced POP is 30.6%. Clinical factors associated with hydronephrosis include degree of anterior or apical POP and diabetes mellitus. Urodynamic factors associated with hydronephrosis include elevated postvoid residuals, larger cystometric capacity, and lower volume at first leak.

Interaction of cowpea mosaic virus nanoparticles with surface vimentin and inflammatory cells in atherosclerotic lesions.

AIMS: Detection of atherosclerosis has generally been limited to the late stages of development, after cardiovascular symptoms present or a clinical event occurs. One possibility for early detection is the use of functionalized nanoparticles. The aim of this study was the early imaging of atherosclerosis using nanoparticles with a natural affinity for inflammatory cells in the lesion. MATERIALS & METHODS: We investigated uptake of cowpea mosaic virus by macrophages and foam cells in vitro and correlated this with vimentin expression. We also examined the ability of cowpea mosaic virus to interact with atherosclerotic lesions in a murine model of atherosclerosis. RESULTS & CONCLUSION: We found that uptake of cowpea mosaic virus is increased in areas of atherosclerotic lesion. This correlated with increased surface vimentin in the lesion compared with nonlesion vasculature. In conclusion, cowpea mosaic virus and its vimentin-binding region holds potential for use as a targeting ligand for early atherosclerotic lesions, and as a probe for detecting upregulation of surface vimentin during inflammation.

Smoldering rheumatoid arthritis: is the Canadian healthcare system neglecting a significant disease population?

OBJECTIVE: To investigate rheumatology practice in Canada with regard to evaluating disease activity status and treatment regimens in patients with rheumatoid arthritis (RA). It was hypothesized that patients with "smoldering" disease activity were not being adequately treated. METHODS: Rheumatologists were invited to participate by the Canadian Rheumatology Association in an audit entitled the Assessment in Rheumatology (AIR) program. From across Canada, 65 rheumatologists participated. One thousand five hundred ninety-six consecutive patients with RA seen in regular clinics were classified according to 4 states of disease activity: remission, controlled adequately, smoldering, and uncontrolled. Demographics (age, sex, geographic region), therapy (nonsteroidal antiinflammatory drugs, disease modifying antirheumatic drugs, biologicals, steroids), joint counts (tender/swollen), comorbidity, and treatment decisions at the time of the visit were recorded. Data were collected at the time of the visit with personal digital assistants (PDA) and aggregated, without personal identifiers, for analysis in SPSS. RESULTS: The majority of patients had "smoldering" (29%) or "uncontrolled" disease (23%), with the remainder in "remission" (15%) or "controlled adequately" (33%) at the time of their visit. Following the appointment, the uncontrolled group had a 100% increase (from 10.4% to 23.4%) in the addition of biological agents; however, there was no significant increase in the rates for those with smoldering disease (19.4% to 20.5%). CONCLUSION: Despite Canada's universal healthcare system, current treatment regimens may not be optimized on the basis of disease activity. A large proportion of patients with RA (29%) seen in Canadian rheumatology practices may be experiencing unnecessary disease for a variety of reasons.

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.

Amiodarone and bepridil inhibit anthrax toxin entry into host cells.

Anthrax lethal toxin is one of the fundamental components believed to be responsible for the virulence of Bacillus anthracis. In order to find novel compounds with anti-lethal toxin properties, we used a cell-based assay to screen a collection of approximately 500 small molecules. Nineteen compounds that blocked lethal toxin-mediated killing of RAW 264.7 macrophages were identified, and we report here on the characterization of the two most potent antitoxic compounds, amiodarone and bepridil. These drugs are used to treat cardiac arrhythmia or angina in humans at doses similar to those that provide protection against lethal toxin in vitro. Our results support a model whereby the antitoxic properties of both drugs result from their ability to block endosomal acidification, thereby blocking toxin entry. Amiodarone was tested in vivo and found to significantly increase survival of lethal toxin-challenged Fischer rats.

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.

A soluble receptor decoy protects rats against anthrax lethal toxin challenge.

Successful postexposure treatment for inhalation anthrax is thought to include neutralization of anthrax toxin. The soluble anthrax toxin receptor/tumor endothelial marker 8 and capillary morphogenesis protein 2 (sATR/TEM8 and sCMG2, respectively) receptor decoys bind to anthrax toxin protective antigen (PA) and compete with cellular receptors for binding. Here, we show that, in a tissue-culture model of intoxication, sCMG2 is a 11.4-fold more potent antitoxin than sATR/TEM8 and that this increased activity corresponds to an approximately 1000-fold higher PA-binding affinity. Stoichiometric concentrations of sCMG2 protect rats against lethal toxin challenge, making sCMG2 one of the most effective anthrax antitoxins described to date.

Macrophage liver X receptor is required for antiatherogenic activity of LXR agonists.

OBJECTIVE: Complications of atherosclerotic cardiovascular disease due to elevated blood cholesterol levels are the major cause of death in the Western world. The liver X receptors, LXRalpha and LXRbeta (LXRs), are ligand-dependent transcription factors that act as cholesterol sensors and coordinately control transcription of genes involved in cholesterol and lipid homeostasis as well as macrophage inflammatory gene expression. LXRs regulate cholesterol balance through activation of ATP-binding cassette transporters that promote cholesterol transport and excretion from the liver, intestine, and macrophage. Although LXR agonists are known to delay progression of atherosclerosis in mouse models, their ability to abrogate preexisting cardiovascular disease by inducing regression and stabilization of established atherosclerotic lesions has not been addressed. METHODS AND RESULTS: We demonstrate that LXR agonist treatment increases ATP-binding cassette transporter expression within preexisting atherosclerotic lesions, resulting in regression of these lesions as well as remodeling from vulnerable to stable lesions and a reduction in macrophage content. Further, using macrophage-selective LXR-deficient mice created by bone marrow transplantation, we provide the first evidence that macrophage LXR expression is necessary for the atheroprotective actions of an LXR agonist. CONCLUSIONS: These data substantiate that drugs targeting macrophage LXR activity may offer therapeutic benefit in the treatment of atherosclerotic cardiovascular disease.

Identification of macrophage liver X receptors as inhibitors of atherosclerosis.

Recent studies have identified the liver X receptors (LXR alpha and LXR beta) as important regulators of cholesterol metabolism and transport. LXRs control transcription of genes critical to a range of biological functions including regulation of high density lipoprotein cholesterol metabolism, hepatic cholesterol catabolism, and intestinal sterol absorption. Although LXR activity has been proposed to be critical for physiologic lipid metabolism and transport, direct evidence linking LXR signaling pathways to the pathogenesis of cardiovascular disease has yet to be established. In this study bone marrow transplantations were used to selectively eliminate macrophage LXR expression in the context of murine models of atherosclerosis. Our results demonstrate that LXRs are endogenous inhibitors of atherogenesis. Additionally, elimination of LXR activity in bone marrow-derived cells mimics many aspects of Tangier disease, a human high density lipoprotein deficiency, including aberrant regulation of cholesterol transporter expression, lipid accumulation in macrophages, splenomegaly, and increased atherosclerosis. These results identify LXRs as targets for intervention in cardiovascular disease.

Lymphotoxin-alpha- and lymphotoxin-beta-deficient mice differ in susceptibility to scrapie: evidence against dendritic cell involvement in neuroinvasion.

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders of humans and animals often initiated by oral intake of an infectious agent. Current evidence suggests that infection occurs initially in the lymphoid tissues and subsequently in the central nervous system (CNS). The identity of infected lymphoid cells remains controversial, but recent studies point to the involvement of both follicular dendritic cells (FDC) and CD11c(+) lymphoid dendritic cells. FDC generation and maintenance in germinal centers is dependent on lymphotoxin alpha (LT-alpha) and LT-beta signaling components. We report here that by the oral route, LT-alpha -/- mice developed scrapie while LT-beta -/- mice did not. Furthermore, LT-alpha -/- mice had a higher incidence and shorter incubation period for developing disease following inoculation than did LT-beta -/- mice. Transplantation of lymphoid tissues from LT-beta -/- mice, which have cervical and mesenteric lymph nodes, into LT-alpha -/- mice, which do not, did not alter the incidence of CNS scrapie. In other studies, a virus that is tropic for and alters functions of CD11c(+) cells did not alter the kinetics of neuroinvasion of scrapie. Our results suggest that neither FDC nor CD11c(+) cells are essential for neuroinvasion after high doses of RML scrapie. Further, it is possible that an as yet unidentified cell found more abundantly in LT-alpha -/- than in LT-beta -/- mice may assist in the amplification of scrapie infection in the periphery and favor susceptibility to CNS disease following peripheral routes of infection.