Relapsed Mpox Keratitis, St. Louis, Missouri, USA.

We describe a case of a 46-year-old man in Missouri, USA, with newly diagnosed advanced HIV and PCR-confirmed mpox keratitis. The keratitis initially resolved after intravenous tecovirimat and penicillin for suspected ocular syphilis coinfection. Despite a confirmatory negative PCR, he developed relapsed, ipsilateral PCR-positive keratitis and severe ocular mpox requiring corneal transplant.

SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.

Exosomes from COVID-19 Patients Carry Tenascin-C and Fibrinogen-ß in Triggering Inflammatory Signals in Cells of Distant Organ.

SARS-CoV-2 infection can cause cytokine storm and may overshoot immunity in humans; however, it remains to be determined whether virus-induced soluble mediators from infected cells are carried by exosomes as vehicles to distant organs and cause tissue damage in COVID-19 patients. We took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and COVID-19 patients. Our results revealed that tenascin-C (TNC) and fibrinogen-ß (FGB) are highly abundant in exosomes from COVID-19 patients' plasma compared with that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via the Nuclear factor-κB (NF-κB) pathway, we examined the status of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-C motif chemokine ligand 5 (CCL5) expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase compared with that from healthy subjects. Together, our results demonstrate that TNC and FGB are transported through plasma exosomes and potentially trigger pro-inflammatory cytokine signaling in cells of distant organ.

Hemoglobin ß93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.

Circulatory Exosomes from COVID-19 Patients Trigger NLRP3 Inflammasome in Endothelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces inflammatory response, cytokine storm, venous thromboembolism, coagulopathy, and multiple organ damage. Resting endothelial cells prevent coagulation, control blood flow, and inhibit inflammation. However, it remains unknown how SARS-CoV-2 induces strong molecular signals in distant cells for immunopathogenesis. In this study, we examined the consequence of human endothelial cells, microvascular endothelial cells (HMEC-1), and liver endothelial cells (TMNK-1) to exosomes isolated from plasma of mild or severe COVID-19 patients. We observed a significant induction of NLRP3, caspase-1, and interleukin-1ß (IL-1ß) mRNA expression in endothelial cells following exposure to exosomes from severe COVID-19 patients compared with that from patients with mild disease or healthy donors. Activation of caspase-1 was noted in the endothelial cell culture medium following exposure to the COVID-19 exosomes. Furthermore, COVID-19 exosomes significantly induced mature IL-1ß secretion in both HMEC-1 and TMNK-1 endothelial cell culture medium. Thus, our results demonstrated for the first time that exosomes from COVID-19 plasma trigger NLRP3 inflammasome in endothelial cells of distant organs resulting in IL-1ß secretion and inflammatory response. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global health problem. Although the vaccine controls infection, understanding the molecular mechanism of pathogenesis will help in developing future therapies. Furthermore, several investigators predicted the involvement of endothelial cell-related inflammation in SARS-CoV-2 infection and using extracellular vesicles as a cargo to carry a drug or vaccine for combating SARS-CoV-2 infection. However, the mechanism by which endothelial cells are inflamed remains unknown. Our present study highlights that exosomes from severe COVID-19 patients can enhance inflammasome activity in distant endothelial cells for augmentation of immunopathogenesis and opens an avenue for developing therapies.

Gabapentin prevalence: clinical and forensic experience in St. Louis, Missouri, USA.

Gabapentin (Neurontin) is an anti-epileptic drug that has had wide off-label prescription use since market release due to presumed negligible abuse potential. However, trends in drug misuse have demonstrated that gabapentin misuse is occurring, particularly in those with a history of opioid misuse. This is concerning, because although gabapentin has no direct ligand activity at opioid receptors, it does potentiate the analgesic effect of opioids, and concurrent use of gabapentin and opioids may increase the risk of respiratory depressive effects of opioids. This study investigates the incidence of gabapentin detected in urine samples collected for clinical drug screening purposes in a local hospital emergency department and in postmortem samples submitted by medical examiners in the St. Louis metropolitan area. The prevalence of gabapentin and co-detected drugs in both populations is contrasted, compared, and discussed. This study found that 30% of urine samples collected from patients with suspected drug intoxication presenting to SSM Health Saint Louis University Hospital, a quaternary care medical center, were positive for gabapentin, and nearly two thirds of those were also positive for oxycodone. Over a 6-month period, the incidence of gabapentin positive postmortem cases increased from 18% to 20%. Nearly all gabapentin positive postmortem cases were also positive for an opioid, the most significant being fentanyl, suggesting that gabapentin misuse may be due to its potentiating effect of opioid drug action. This study also highlights the limited utility of immunoassay-based urine drug screens.

Momordicine-I, a Bitter Melon Bioactive Metabolite, Displays Anti-Tumor Activity in Head and Neck Cancer Involving c-Met and Downstream Signaling.

Head and neck cancer (HNC) is one of the most aggressive cancers, and treatments are quite challenging due to the difficulty in early diagnosis, lack of effective chemotherapeutic drugs, adverse side effects and therapy resistance. We identified momordicine-I (M-I), a bioactive secondary metabolite in bitter melon (Momordica charantia), by performing liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) analysis. M-I inhibited human HNC cell (JHU022, JHU029, Cal27) viability in a dose-dependent manner without an apparent toxic effect on normal oral keratinocytes. Mechanistic studies showed that M-I inhibited c-Met and its downstream signaling molecules c-Myc, survivin, and cyclin D1 through the inactivation of STAT3 in HNC cells. We further observed that M-I was non-toxic and stable in mouse (male C57Bl/6) blood, and a favorable pharmacokinetics profile was observed after IP administration. M-I treatment reduced HNC xenograft tumor growth in nude mice and inhibited c-Met and downstream signaling. Thus, M-I has potential therapeutic implications against HNC.

Professional Certification in Point-of-Care Testing.

Professional certification is affirmation and documentation that the certified individual has the knowledge, training, and skills necessary to practice some aspect of medicine or other profession. Herein is a description of the genesis of a professional certification in point of care testing (POCT), inclusive of rationale and goals. A distinction between professional certification and certificate training programs is made. Details regarding eligibility to sit for the board exam are provided along with a list exam content areas. Finally, successes of this professional certification program are highlighted.

Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation.

Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.

Hydrogen sulfide mediates the vasoactivity of garlic.

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H(2)S), an endogenous cardioprotective vascular cell signaling molecule. This H(2)S production, measured in real time by a novel polarographic H(2)S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H(2)S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the alpha carbon of the allyl substituent, thereby forming a hydropolysulfide (RS(n)H), a key intermediate during the formation of H(2)S. Organic polysulfides (R-S(n)-R'; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RS(n)H and H(2)S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H(2)S. The vasoactivity of garlic compounds is synchronous with H(2)S production, and their potency to mediate relaxation increases with H(2)S yield, strongly supporting our hypothesis that H(2)S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H(2)S can be used to standardize garlic dietary supplements.

Evaluation of a Novel Light Scattering Methodology for the Detection of Pathogenic Bacteria in Urine.

BACKGROUND: Urine culture, the gold standard for detecting and identifying bacteria in urine, is one of the highest volume tests in many microbiology laboratories. The inability to accurately predict which patients would benefit from culture leads not only to monopolization of laboratory resources, but also to unnecessary antimicrobial exposure as patients receive empirical treatment for suspected or presumed urinary tract infections (UTI) while awaiting culture results. A common approach to decrease unnecessary urine culture is screening samples using urinalysis (UA) parameters to determine those that should proceed to culture (reflex). In this study, we compared the performance of a novel uropathogen detection method to urinalysis for purposes of UTI screening. METHODS: Urine specimens submitted for culture (n = 194) were evaluated by urinalysis and a novel light scattering device (BacterioScan 216Dx UTI System) capable of detecting the presence of bacteria in urine. Sensitivity and specificity for prediction of a positive urine culture by UA and 216Dx were determined relative to urine culture results. A positive urine culture was defined as growth in culture of one or two uropathogens at concentrations of ≥50,000 CFU/mL. RESULTS: 194 urine samples were evaluated by UA, 216Dx, and urine culture. The 216Dx demonstrated a 100% [95%CI: 88.43%-100.0%] sensitivity and 81.71% [95%CI: 74.93%-87.30%] specificity for the detection of bacteriuria, vs UA with a sensitivity of 86.67% [95%CI 69.28%-96.24%] and specificity of 71.95% [95%CI: 64.41%-78.68%] when compared to urine culture (diagnostic reference method). CONCLUSIONS: BacterioScan allows for an alternative method of screening with satisfactory sensitivity and improved specificity that may facilitate a reduction of unnecessary cultures. Additional studies are required to determine if a concomitant decrease in inappropriate antibiotic use can be realized with the 216Dx technology.

The 2018 AACC/SYCL PhD Clinical Chemist Compensation Survey.

BACKGROUND: Doctoral level board-certified clinical chemists play an invaluable role in many facets of laboratory medicine and healthcare. However, information concerning their total compensation is sparse. CONTENT: A confidential self-reported compensation survey was conducted by the American Association for Clinical Chemistry's Society for Young Clinical Laboratorians (AACC SYCL) Core Committee from April 1 to April 17, 2018. Respondents provided information on geographic location, employment sector, gender, and years of experience to account for the influence of these variables on compensation. There were 199 respondents in total from the United States and Canada, however, only respondents employed in the United States with an earned doctoral degree and certification by the American Board of Clinical Chemistry (n = 133), were included in the full analysis. In comparison to compensation reported in AACC SYCL salary surveys conducted in 2010 and 2013, early career median salaries are trending upwards after correction for inflation. SUMMARY: This survey is the first to collect the gender of respondents, and identify a pay gap for some geographic groups. However, this gap could be due in part to a difference in the years of experience, since males were highly represented in the group with >20 years of experience (25 out of 35, 71%). Future studies on compensation trends within clinical chemistry that do not rely on self-report are needed to ensure accuracy and completeness of the dataset.

Bitter melon extract inhibits breast cancer growth in preclinical model by inducing autophagic cell death.

Breast cancer is a major public health problem worldwide in women and current therapeutic strategies are not adequately effective for this deadly disease. We have previously shown the anti-proliferative activity of bitter melon extract (BME) in breast cancer cells. In this study, we observed that BME treatment induces autophagosome-bound Long chain 3 (LC3)-B and accumulates protein p62/SQSTM1 (p62) in breast cancer cells. Additionally, we observed that BME treatment in breast cancer cells increases phospho-AMPK expression and inhibits the mTOR/Akt signaling pathway. Subsequently, we demonstrated that BME feeding effectively inhibited breast cancer growth in syngeneic and xenograft mouse models. Further, we observed the increased p62 accumulation, induction of autophagy and apoptotic cell death in tumors from BME-fed animals. Taken together, our results demonstrate that BME treatment inhibits breast tumor growth, and this anti-tumor activity in breast cancer is, in part, mediated by induction of autophagy and modulation of the AMPK/mTOR pathway. The antitumor activity of BME by oral feeding in breast cancer models suggested the high potential for a clinical application.

Cigarette smoke-induced urothelial cell damage: potential role of platelet-activating factor.

Cigarette smoking is an environmental risk factor associated with a variety of pathologies including cardiovascular disease, inflammation, and cancer development. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory bladder disease with multiple etiological contributors and risk factors associated with its development, including cigarette smoking. Previously, we determined that cigarette smoking was associated with bladder wall accumulation of platelet activating factor (PAF), a potent inflammatory mediator that facilitates transendothelial cell migration of inflammatory cells from the circulation. PAF has been shown to reduce expression of tight junctional proteins which could ultimately lead to increased urothelial cell permeability. In this study, we observed that cigarette smoke extract (CSE) treatment of human urothelial cells increases PAF production and PAF receptor expression and reduces wound healing ability. After exposure to cigarette smoke for 6 months, wild-type C57BL/6 mice displayed urothelial thinning and destruction which was not detected in iPLA2ß-/- (enzyme responsible for PAF production) animals. We also detected increased urinary PAF concentration in IC/BPS patients when compared to controls, with an even greater increase in urinary PAF concentration in smokers with IC/BPS These data indicate that cigarette smoking is associated with urothelial cell damage that may be a result of increased PAF-PAF receptor interaction. Inhibition of iPLA2ß activity or blocking of the PAF-PAF receptor interaction could serve as a potential therapeutic target for managing cigarette smoke-induced bladder damage.

Assessing NO-dependent vasodilatation using vessel bioassays at defined oxygen tensions.

Results from vessel bioassays have provided the foundation for much of our understanding of the mechanisms that control vascular homeostasis and blood flow. The seminal observations that led to the discovery that nitric oxide (NO) is a critical mediator of vascular relaxation were made with the use of such methodology, and many studies have used NO-dependent vessel relaxation as an experimental readout for understanding mechanisms that regulate vascular NO function. Studies have coupled controlling oxygen tensions within vessel bioassay chambers to begin to understand how oxygen-specifically hypoxia-regulate NO function, and this context has identified red cells-specifically hemoglobin within-as critical modulators. Alone, vessel bioassays or measuring oxygen partial pressures (pO2) is relatively straightforward, but the combination necessitates consideration of several factors. We use the example of deoxygenated red cells/hemoglobin-dependent potentiation of nitrite-dependent dilation to illustrate the salient factors that are critical to consider in designing and interpreting experiments aimed at understanding the interplay between oxygen and NO function in the vasculature.