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Objectives: To determine the occurrence rate and impact on patient outcomes of probiotic-associated central venous catheter bloodstream infections in the ICU., Design: Retrospective observational cohort study., Setting: The cohort was gathered using HCA Healthcare's data warehouse., Patients: Adult patients with central venous catheters in the ICU., Interventions: None., Measurements and Main Results: Blood culture data were used to determine whether an infection had occurred with an organism contained in an administered probiotic. Eighty-six probiotic-associated central venous catheter bloodstream infections were identified among the 23,015 patient cohort who received probiotics (0.37%). The number needed to harm was 270. Zero infections were found in the cohort that did not receive probiotics. Patients who contracted a probiotic infection had increased mortality (odds ratio, 2.23; 1.30-3.71; p < 0.01). Powder formulations had an increased rate of infection compared with nonpowder formulations (0.76% vs 0.33%; odds ratio, 2.03; 1.05-3.95; p = 0.04)., Conclusions: Probiotic administration is associated with a substantial rate of probiotic-associated bloodstream infection in ICU patients with central venous catheters in place. Probiotic-associated bloodstream infections result in significantly increased mortality. Powder formulations cause bloodstream infections more frequently than nonpowder formulations. In ICU patients with central venous catheters, the risks of probiotic-associated central venous catheter bloodstream infection and death outweigh any potential benefits of probiotic administration., Competing Interests: Mr. Cornett received funding from HCA Healthcare. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2023 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

Herein, we present a unique case of a Coombs-negative, steroid-refractory autoimmune hemolytic anemia (AIHA) complicated by pseudoreticulopenia, describe its clinical presentation, histopathologic findings, and management, and review the salient literature. Coombs-negative, steroid-refractory AIHAs represent fewer than 1% of all AIHAs. Diagnosis of the disease is difficult and often delayed due to the pursuit of alternate diagnoses following a negative Coombs test. However, when suspicion remains high for an autoimmune process, the super-Coombs test may be utilized for the diagnosis of AIHA that the traditional Coombs test fails to detect. A majority of cases respond to rituximab as the indicated second-line therapy, but delays in diagnosis and subsequent treatment may increase morbidity. Reticulopenia may be associated with AIHAs secondary to bone marrow dysfunction, but this patient had a normal function marrow confirmed on biopsy. Indeed, reticulopenia in this case was a diagnostic conundrum that further obscured the diagnosis and delayed treatment. Ultimately, reticulopenia was determined to be pseudoreticulopenia secondary to an alteration in the maturation of the erythroid lineage due to an independent, newly diagnosed pernicious anemia. The interaction of these multiple coexisting disease processes is not previously described in the literature. Increased physician awareness of steroid-refractory, Coombs-negative AIHA, and the development of pseudoreticulopenia as a laboratory finding in pernicious anemia may help to improve patient outcomes., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Scott Mayer et al.)

Background: Langerhans cell histiocytosis (LCH) of the temporal bone is an uncommon disease that primarily affects the pediatric population; fewer than 40 adult cases have been reported in the literature. We present a rare case of LCH of the temporal bone in an adult patient and describe its clinical presentation, histopathologic findings, and management. Case Report : A 21-year-old male presented to the emergency department with progressively worsening right-sided ear pain refractory to outpatient oral antibiotics. Physical examination revealed mastoid tenderness and decreased right-sided hearing. Computed tomography (CT) scan suggested coalescent mastoiditis; the patient responded to inpatient antibiotics and was discharged. He returned 9 days later with persistent symptoms. Repeat CT scan revealed an osteolytic lesion on the temporal bone, and the patient was indicated for surgery. Intraoperative histology was consistent with LCH. Subsequent surveillance magnetic resonance imaging (MRI) suggested persistence of disease, and the patient responded to a course of radiation. Three months following radiotherapy, surveillance MRI and positron emission tomography scans revealed no evidence of recurrent disease. Conclusion: Diagnosis of LCH of the temporal bone is frequently delayed because of misdiagnosis of more common otologic diseases, including otitis media, otitis externa, and mastoiditis. The clinician's index of suspicion for LCH should be high if imaging reveals an osteolytic defect of the temporal bone; confirmation is via immunohistostaining of biopsy samples. The majority of cases respond to surgery, radiation, chemotherapy, or combination therapy, but delays in diagnosis and treatment may increase morbidity. Increased physician awareness of LCH of the temporal bone, particularly among adults, may help to improve patient outcomes., (©2020 by the author(s); Creative Commons Attribution License (CC BY).)

Background: Alcohol-related motor vehicle collisions (AR-MVCs) account for ~30% of all US traffic fatalities. Ride-sharing services (RSS) have existed since 2010, but few studies to date have investigated their impact on AR-MVCs. We hypothesized that the availability of RSS would be correlated with a decrease in AR-MVCs at an urban Level I trauma center., Methods: A retrospective chart review was conducted of all AR-MVC trauma activations at a Level I trauma center from 2012 to 2018. Additional data were gathered from regional governmental traffic and law enforcement databases, including crash incidence, fatalities, and demographics. Data were compared pre- and post-RSS and analyzed using an unpaired t test with p less than 0.05 considered significant., Results: There were 1,474 patients in AR-MVCs during the study period. There was a significant decrease in the annual average proportion of MVCs that were AR-MVCs pre- vs. post-RSS (39% vs. 29%, p = 0.02) as well as a decrease in the average annual incidence of fatal AR-MVCs (11.6 vs. 5, p = 0.02). Subset analysis showed a decrease in AR-MVC incidence in 18- to 29-year-olds (12.7% vs. 7.5%; p = 0.03), which was also demonstrated by data from a local law enforcement database. Availability of RSS was also correlated with a decreased proportion of nighttime AR-MVCs (14.7% vs. 7.6%, p = 0.03) and decreased number of driving while intoxicated (1198.0 ± 78.5 vs. 612.8 ± 137.6, p = <0.01)., Conclusion: We found that the incidence of both total AR-MVCs and fatal AR-MVCs presenting to our trauma center decreased after the introduction of RSS. Ride-sharing services may play a role in preventing AR-MVCs. Further research is needed to correlate AR-MVC incidence with granular proprietary RSS usage data and to account for any confounding factors. Future studies may identify ways to better utilize RSS availability as a targeted intervention for certain demographic groups to prevent AR-MVCs., Level of Evidence: Therapeutic/Care Management, Level IV.

Background: Signs of inflammation are commonly encountered during endoscopic examination of the Eustachian tube (ET) region. The clinical applicability of these findings may be enhanced by use of a standardized assessment score., Methods: Digital video recordings were obtained of 50 nasal endoscopy examinations of the nasopharyngeal portion of the ET. Four fellowship-trained rhinologists independently reviewed the videos with regard to specific physical findings: edema of the ET torus, erythema of the ET torus, exudate at the ET orifice, and presence of tubal tonsil. Scoring of this Endoscopic Evaluation of the Eustachian Tube (3ET) was reported using both 2-point and 3-point scales. Each reviewer repeated the scoring at a 10-day interval. Interrater and intrarater agreement were calculated for each item and the total scores., Results: Interrater and intrarater agreement were greater for the 3-point scale than the 2-point scale. Interrater agreement for overall instrument using the 3-point scale was in the "acceptable" range for Krippendorff's alpha on both the first trial (0.6922) and second trial (0.7238). Intrarater agreement was generally "excellent" for individual items as well as the overall instrument., Conclusion: The 3ET comprising these 4 physical findings has acceptable interrater and intrarater reliability, and may be applied to future clinical studies of ET function and disease., (© 2018 ARS-AAOA, LLC.)

Ion channels have provided a diverse set of therapeutic targets across all areas of the pharmaceutical industry. Many companies are pursuing this unique class of targets for areas of unmet medical need such as neuropathic and inflammatory pains. In the past, focused library screening sets had been designed for CNS and kinase targets. Our investigations were aimed at creating a similar dynamic screening set enriched for compounds targeting ion channels to aid screening efforts of this important class of targets. The key advantages of this approach for ion channel targets would be: (1) to identify tool compounds for novel targets and assist in assay validation, (2) to serve as a focused screen for non-384-well adaptable targets, and (3) to jump start a particular program, that is, catch-up to competition for validated, well-known targets.

The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the well-to-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 +/- 1500 FLIPR(3) relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 microM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r(2) = 0.56) between the respective IC(50)s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds.

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimer's disease.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker currents in neurons (I(h)) and cardiac (I(f)) cells. As such, the identification and characterization of novel blockers of HCN channels is important to enable the dissection of their function in vivo. Using a new IonWorks HT electrophysiology assay with human HCN1 and HCN4 expressed stably in cell lines, four HCN channel blockers are characterized. Two blockers known for their activity at opioid/Ca(2+) channels and K(+) channels, loperamide and CP-339,818 (respectively), are described to block HCN1 more potently than HCN4. The known HCN blocker ZD7288 was also found to be more selective for HCN1 over HCN4, while the HCN blocker DK-AH269 was equipotent on HCN4 and HCN1. Partial replacement of the intracellular Cl(-) with gluconate reduced the potency on both channels, but to varying degrees. For both HCN1 and HCN4, ZD7288 was most sensitive in lower Cl(-) solutions, while the potency of loperamide was not affected by the differing solutions. The block of HCN1 for all compounds was voltage-dependent, being relieved at more negative potentials. The voltage-dependent, Cl(-) dependent, HCN1 preferring compounds described here elaborate on the current known pharmacology of HCN channels and may help provide novel tools and chemical starting points for the investigation of HCN channel function in natively expressing systems.

PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.

An asymmetric synthesis of alpha-amino acids with novel beta-branched side chains has been implemented. The syntheses feature a p-toluenesulfinylimine induced chiral Strecker approach and were found to be applicable to the introduction of both aliphatic and aromatic beta-branched sidechains for preparation of previously unknown alpha-amino acids.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited I(h) channels in rat DRG neurons. Loperamide blocked I(h) in a concentration-dependent manner, with an IC(50) = 4.9 +/- 0.6 and 11.0 +/- 0.5 microM for large- and small-diameter neurons, respectively. Loperamide-induced I(h) inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V(1/2) for I(h) steady-state activation. Loperamide block of I(h) was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at -60 mV to 4% at -120 mV in the presence of 3.7 microM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of I(h). Inclusion of 10 microM loperamide into the recording pipette did not affect I(h) voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible I(h) inhibition. The observed loperamide-induced I(h) inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse I(h) inhibition. Therefore we suggest that loperamide inhibits I(h) by direct binding to the extracellular region of the channel. Because I(h) channels are involved in pain processing, loperamide-induced inhibition of I(h) channels could provide an additional molecular mechanism for its analgesic action.

Alzheimer's disease (AD) is a debilitating disease widely thought to be associated with the accumulation of beta amyloid (Abeta) in the brain. Inhibition of gamma-secretase, one of the enzymes responsible for Abeta production, may be a useful strategy for the treatment of AD. Described below is a series of gamma-secretase inhibitors designed from a scaffold identified by a ROCS [J. Comput. Chem.1996, 17, 1653] search of the corporate database.

In our investigations to synthesize inhibitors of smooth muscle cell (SMC) proliferation, compound 6a displayed submicromolar activity in in vitro antiproliferative assays and reduced intimal thickening using a rat balloon angioplasty model via i.v. administration. In order to identify analogs that could be administered orally, the chemical instability of the C-4',6' acetal of compound 6a was addressed. Several novel variants with increased acid stability and comparable in vitro activity were prepared.

The didemnin class of biologically active cyclodepsipeptides, isolated from a marine tunicate, has shown antitumor, antiviral, and immunosuppressive activities. Synthetic studies were undertaken to prepare a modified analog of one of the most potent congeners, didemnin B (1). The side chain of the isostatine unit was tethered into the macrocycle viaa cyclohexane ring in order to provide a more rigid conformation and determine the importance of this unit in bioactive compounds. This modification created a new macrocycle core and generated a diastereomeric mixture of a constrained analog of didemnin B (2).