Validated model of platelet slip at stenosis and device surfaces.

Platelets are central to thrombosis. However, it is unknown whether platelets slip at vascular or device surfaces. The presence of platelet slip at a surface would interrupt physical contact between the platelet and that surface, and therefore diminish adhesion and thrombosis. Unfortunately, no existing technology can directly measure platelet slip in a biological environment. The objective of this study was to explore whether microspheres-modeling platelets-slip at different vascular and device surfaces in an acrylic scaled-up model coronary artery. The microspheres (3.12 µm diameter) were suspended in a transparent glycerol/water experimental fluid, which flowed continuously at Reynolds numbers typical of coronary flow (200-400) through the model artery. We placed a series of axisymmetric acrylic stenoses (cross-sectional area reduction [CSAr], 20-90%) into the model artery, both without and with a central cylinder present (modeling a percutaneous interventional guide wire, and with a scaled-up Doppler catheter mounted upstream). We used laser Doppler velocimetry (LDV) to measure microsphere velocities within, proximal and distal to each stenosis, and compared to computer simulations of fluid flow with no-slip. For validation, we replaced the acrylic with paraffin stenoses (more biologically relevant from a surface roughness perspective) and then analyzed the signal recorded by the scaled-up Doppler catheter. Using the LDV, we identified progressive microsphere slip proportional to CSAr inside entrances for stenoses ≥60% and ≥40% without and with cylinder present, respectively. Additionally, microsphere slip occurred universally along the cylinder surface. Computer simulations indicated increased fluid shear rates (velocity gradients) at these particular locations, and logistic regression analysis comparing microsphere slip with fluid shear rate resulted in a c-index of 0.989 at a cut-point fluid shear rate of (10.61 [cm-1]×mean velocity [cm×sec-1]). Moreover, the presence of the cylinder caused disordering of microsphere shear rates distal to higher grade stenoses, indicating a disturbance in their flow. Finally, despite lower precision, the signal recorded by the scaled-up Doppler catheter nonetheless indicated slip at the entry into and at most locations distal to the 90% stenosis. Our validated model establishes proof of concept for platelet slip, and platelet slip explains several important basic and clinical observations. If technological advances allow confirmation in a true biologic environment, then our results will likely influence the development of shear-dependent antiplatelet drugs. Also, adding shear rate information, our results provide a direct experimental fluid dynamic foundation for antiplatelet-focused antithrombotic therapy during coronary interventions directed towards higher grade atherosclerotic stenoses.

Safely reducing haemodialysis frequency during the COVID-19 pandemic.

BACKGROUND: Patients undergoing haemodialysis (HD) are at higher risk of developing worse outcomes if they contract COVID-19. In our renal service we reduced HD frequency from thrice to twice-weekly in selected patients with the primary aim of reducing COVID 19 exposure and transmission between HD patients. METHODS: Dialysis unit nephrologists identified 166 suitable patients (38.4% of our HD population) to temporarily convert to twice-weekly haemodialysis immediately prior to the peak of the COVID-19 pandemic in our area. Changes in pre-dialysis weight, systolic blood pressure (SBP) and biochemistry were recorded weekly throughout the 4-week project. Hyperkalaemic patients (serum potassium > 6.0 mmol/L) were treated with a potassium binder, sodium bicarbonate and received responsive dietary advice. RESULTS: There were 12 deaths (5 due to COVID-19) in the HD population, 6 of which were in the twice weekly HD group; no deaths were definitively associated with change of dialysis protocol. A further 19 patients were either hospitalised and/or developed COVID-19 and thus transferred back to thrice weekly dialysis as per protocol. 113 (68.1%) were still receiving twice-weekly HD by the end of the 4-week project. Indications for transfer back to thrice weekly were; fluid overload (19), persistent hyperkalaemia (4), patient request (4) and compliance (1). There were statistically significant increases in SBP and pre-dialysis potassium during the project. CONCLUSIONS: Short term conversion of a large but selected HD population to twice-weekly dialysis sessions was possible and safe. This approach could help mitigate COVID-19 transmission amongst dialysis patients in centres with similar organisational pressures.

Identification of multiple species and subpopulations among Australian clinical Sporothrix isolates using whole genome sequencing.

Whole genome sequencing (WGS) was used to demonstrate the wide genetic variability within Sporothrix schenckii sensu lato and establish that there are two main species of Sporothrix within Australian clinical isolates-S. schenckii sensu stricto and Sporothrix globosa. We also demonstrated southwest Western Australia contained genetically similar S. schenckii ss strains that are distinct from strains isolated in the eastern and northern states of Australia. Some genetic clustering by region was also noted for northern NSW, Queensland, and Northern Territory. Phylogenetic analysis of WGS data provided greater phylogenetic resolution compared to analysis of the calmodulin gene alone.

Retinal pigment epithelium and microglia express the CD5 antigen-like protein, a novel autoantigen in age-related macular degeneration.

We report on a novel autoantigen expressed in human macular tissues, identified following an initial Western blot (WB)-based screening of sera from subjects with age-related macular degeneration (AMD) for circulating auto-antibodies (AAbs) recognizing macular antigens. Immunoprecipitation, 2D-gel electrophoresis (2D-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), direct enzyme-linked immunosorbent assays (ELISA), WBs, immunohistochemistry (IHC), human primary and ARPE-19 immortalized cell cultures were used to characterize this novel antigen. An approximately 40-kDa autoantigen in AMD was identified as the scavenger receptor CD5 antigen-like protein (CD5L), also known as apoptosis inhibitor of macrophage (AIM). CD5L/AIM was localized to human RPE by IHC and WB methods and to retinal microglial cells by IHC. ELISAs with recombinant CD5L/AIM on a subset of AMD sera showed a nearly 2-fold higher anti-CD5L/AIM reactivity in AMD vs. Control sera (p = 0.000007). Reactivity ≥0.4 was associated with 18-fold higher odds of having AMD (χ2 = 21.42, p = 0.00063). Circulating CD5L/AIM levels were also nearly 2-fold higher in AMD sera compared to controls (p = 0.0052). The discovery of CD5L/AIM expression in the RPE and in retinal microglial cells adds to the known immunomodulatory roles of these cells in the retina. The discovery of AAbs recognizing CD5L/AIM identifies a possible novel disease biomarker and suggest a potential role for CD5L/AIM in the pathogenesis of AMD in situ. The possible mechanisms via which anti-CD5L/AIM AAbs may contribute to AMD pathogenesis are discussed. In particular, since CD5L is known to stimulate autophagy and to participate in oxidized LDL uptake in macrophages, we propose that anti-CD5L/AIM auto-antibodies may play a role in drusen biogenesis and inflammatory RPE damage in AMD.

The clinical significance of hyperkalaemia-associated repolarization abnormalities in end-stage renal disease.

BACKGROUND: Hyperkalaemia is a common potentially fatal complication of chronic kidney disease (CKD). It may manifest as electrocardiogram (ECG) changes, the earliest of which is T-wave 'tenting'. However, this occurs in less than half of episodes of hyperkalaemia. The aim of this study was to determine what other clinical features relate to the probability of T-wave tenting; and if there is a longer-term survival difference between patients who develop tenting and those who do not. METHOD: One hundred and forty-five patients with end-stage renal disease who had standard 12-lead ECG and concurrent serum potassium measurement were enrolled. The presence of tenting and the ratio of the amplitude of the tallest precordial T-wave and R-wave were determined (T:R). RESULTS: Tenting was as common in normal range serum potassium as hyperkalaemia (33 versus 31%) and less common than in left ventricular hypertrophy (44%). T:R was less sensitive (24 versus 33%) but more specific (85 versus 67%) than tenting at correctly identifying hyperkalaemia ≥ 6.0 mmol/L. Tenting became less common with increasing age. Dialysis patients were more likely to show increased T:R that pre-dialysis Stage 5 CKD. Elevated T:R was not associated with worse cardiovascular outcome but was associated with increased risk of sudden death over a mean follow-up of 3.8 years (hazard ratio = 8.3, P = 0.021). CONCLUSIONS: The reason for the variability in T-wave changes is not clear. The ratio of precordial T-wave to R-wave amplitude is a more specific measure than tenting but both are poorly sensitive at detecting hyperkalaemia. The greater risk for sudden death may represent a susceptibility to cardiac arrhythmia during repolarization.

Illness severity in community-onset invasive Staphylococcus aureus infection and the presence of virulence genes.

BACKGROUND: It is uncertain whether particular clones causing invasive community-onset methicillin-resistant and methicillin-sensitive Staphylococcus aureus (cMRSA/cMSSA) infection differ in virulence. METHODS: Invasive cMRSA and cMSSA cases were prospectively identified. Principal component analysis was used to derive an illness severity score (ISS) from clinical data, including 30-day mortality, requirement for intensive hospital support, the presence of bloodstream infection, and hospital length of stay. The mean ISS for each S. aureus clone (based on MLST) was compared with its DNA microarray-based genotype. RESULTS: Fifty-seven cMRSA and 50 cMSSA infections were analyzed. Ten clones caused 82 (77%) of these infections and had an ISS calculated. The enterotoxin gene cluster (egc) and the collagen adhesin (cna) gene were found in 4 of the 5 highest-ranked clones (ST47-MSSA, ST30-MRSA-IV[2B], ST45-MSSA, and ST22-MRSA-IV[2B]) compared with none and 1 of the lowest 5 ranked clones, respectively. cMSSA clones caused more severe infection than cMRSA clones. The lukF/lukS Panton-Valentine leukocidin (PVL) genes did not directly correlate with the ISS, being present in the second, fourth, and 10th most virulent clones. CONCLUSIONS: The clinical severity of invasive cMRSA and cMSSA infection is likely to be attributable to the isolates' entire genotype rather than a single putative virulence determinant such as PVL.

Antihypertensive prescribing patterns in non-dialysis dependent chronic kidney disease: Findings from the Salford Kidney Study.

BACKGROUND: Hypertension is commonly observed in patients living with chronic kidney disease (CKD). Finding an optimal treatment regime remains challenging due to the complex bidirectional cause-and-effect relationship between hypertension and CKD. There remains variability in antihypertensive treatment practices. AIM: To analyze data from the Salford Kidney Study database in relation to antihypertensive prescribing patterns amongst CKD patients. METHODS: The Salford Kidney Study is an ongoing prospective study that has been recruiting CKD patients since 2002. All patients are followed up annually, and their medical records including the list of medications are updated until they reach study endpoints [starting on renal replacement therapy or reaching estimated glomerular filtration rate (eGFR) expressed as mL/min/1.73 m2 ≤ 10 mL/min/1.73 m2, or the last follow-up date, or data lock on December 31, 2021, or death]. Data on antihypertensive prescription practices in correspondence to baseline eGFR, urine albumin-creatinine ratio, primary CKD aetiology, and cardiovascular disease were evaluated. Associations between patients who were prescribed three or more antihypertensive agents and their clinical outcomes were studied by Cox regression analysis. Kaplan-Meier analysis demonstrated differences in survival probabilities. RESULTS: Three thousand two hundred and thirty non-dialysis-dependent CKD patients with data collected between October 2002 and December 2019 were included. The median age was 65 years. A greater proportion of patients were taking three or more antihypertensive agents with advancing CKD stages (53% of eGFR ≤ 15 mL/min/1.73 m2 vs 26% of eGFR ≥ 60 mL/min/1.73 m2, P < 0.001). An increased number of patients receiving more classes of antihypertensive agents was observed as the urine albumin-creatinine ratio category increased (category A3: 62% vs category A1: 40%, P < 0.001), with the upward trends particularly noticeable in the number of individuals prescribed renin angiotensin system blockers. The prescription of three or more antihypertensive agents was associated with all-cause mortality, independent of blood pressure control (hazard ratio: 1.15; 95% confidence interval: 1.04-1.27, P = 0.006). Kaplan-Meier analysis illustrated significant differences in survival outcomes between patients with three or more and those with less than three antihypertensive agents prescribed (log-rank, P < 0.001). CONCLUSION: Antihypertensive prescribing patterns in the Salford Kidney Study based on CKD stage were consistent with expectations from the current United Kingdom National Institute of Health and Care Excellence guideline algorithm. Outcomes were poorer in patients with poor blood pressure control despite being on multiple antihypertensive agents. Continued research is required to bridge remaining variations in hypertension treatment practices worldwide.

IGF-I increases the expression of fibronectin by Nox4-dependent Akt phosphorylation in renal tubular epithelial cells.

Extracellular matrix accumulation contributes to the progression of chronic kidney disease. Many growth factors including insulin-like growth factor-I (IGF-I) enhance matrix protein accumulation. Proximal tubular epithelial cells (PTCs) synthesize matrix proteins. NADPH oxidases are major sources of reactive oxygen species (ROS), important signaling molecules that mediate biological responses in a variety of cells and tissue. We investigated the mechanism by which IGF-I regulates fibronectin accumulation in PTCs and the role of a potential redox-dependent signaling pathway. IGF-I induces an increase in NADPH-dependent superoxide generation, enhances the release of hydrogen peroxide, and increases the expression of NADPH oxidase 4 (Nox4) in PTCs. IGF-I also stimulates phosphorylation of Akt, and inhibition of Akt or its upstream activator phosphatidylinositol 3-kinase attenuates IGF-I-induced fibronectin accumulation. Expression of dominant negative Akt also inhibits IGF-I-induced expression of fibronectin, indicating a role for this kinase in fibronectin accumulation. Expression of dominant negative adenovirus Nox4 inhibits IGF-I-induced NADPH oxidase activity, Akt phosphorylation, and fibronectin protein expression. Moreover, transfection of small interfering RNA targeting Nox4 decreases Nox4 protein expression and blocks IGF-I-induced Akt phosphorylation and the increase in fibronectin, placing Nox4 and ROS upstream of Akt signaling pathway. To confirm the role of Nox4, PTCs were infected with adenovirus construct expressing wild-type Nox4. Ad-Nox4, but not control Ad-green fluorescent protein, upregulated Nox4 expression and increased NADPH oxidase activity as well as fibronectin expression. Taken together, these results provide the first evidence for a role of Nox4 in IGF-I-induced Akt phosphorylation and fibronectin expression in tubular epithelial cells.

Body mass index has no effect on rate of progression of chronic kidney disease in non-diabetic subjects.

BACKGROUND: Chronic kidney disease (CKD) is increasingly prevalent worldwide. Furthermore, obesity is now a global problem with major health implications. There is a clear association between obesity and the development of CKD but it is not known whether obesity is a risk factor for the progression of pre-existing kidney disease. We examined the relationship between the body mass index (BMI) and the rate of progression of CKD in non-diabetic adults. METHODS: The Chronic Renal Insufficiency Standards Implementation Study (CRISIS) is a prospective observational study in a predominantly white population in Greater Manchester. From the CRISIS database, we assessed rate of progression of CKD in 499 adults attending the hospital. Baseline measurements including BMI were obtained and estimated glomerular filtration rate (eGFR) was monitored. The rate of deterioration of eGFR was derived over time, defined as ΔeGFR (mL/min/1.73 m2/year) and assessed using univariate analysis of variance. RESULTS: In the groups as a whole, no relationship between BMI and ΔeGFR was shown. Dividing the subjects into obese (BMI≥30) and non-obese (BMI<30) groups and further breakdown into CKD stages 3, 4 and 5, also showed no relationship between BMI and ΔeGFR. Univariate analysis of variance was used. CONCLUSIONS: Neither BMI as a continuous variable nor obesity (BMI≥30) as a categorical variable was associated with an increased rate of progression of existing CKD in this predominantly white population.

How accurately do nephrologists predict the need for dialysis within one year?

BACKGROUND/AIMS: Knowing when patients with chronic kidney disease will need dialysis can improve patient counselling and timing of vascular access. We aimed to assess the accuracy of clinician judgement in predicting the need for dialysis within 12 months. METHODS: We asked the nephrologists in a dedicated pre-dialysis clinic to predict the time until initiation of dialysis for patients. We compared predicted with actual time to dialysis and the accuracy of predictions made by different grades of clinician. Multivariate logistic regression compared clinical parameters that correlated with predicted and actual time to dialysis. RESULTS: One hundred and eighty-four patients were included. The sensitivity of clinician judgement as a predictor of dialysis within 12 months was 95% and the specificity was 62%. Consultants were correct in 71% of cases and trainees in 68% of cases. Estimated glomerular filtration rate (eGFR) was the only independent correlate of predicted time to dialysis [odds ratio (OR) = 1.6 per 1 ml/min/1.73 m(2) reduction, p < 0.001]. eGFR was also associated with actual time to dialysis (OR = 1.6 per 1 ml/min/1.73 m(2), p < 0.001) along with age (OR = 0.94 per year increase, p = 0.005) and itch (OR = 3.7, p = 0.048). CONCLUSION: Clinical judgement is sensitive but not specific in predicting the need for dialysis. Educating the clinicians may improve the specificity of judgement and improve the accuracy of prognostic information given to patients.

The NADPH oxidase subunit p22phox inhibits the function of the tumor suppressor protein tuberin.

Mutations in the von Hippel-Lindau (VHL) gene give rise to renal cell carcinoma. Reactive oxygen species, generated by Nox oxidases, are involved in tumorigenesis. We have previously demonstrated that in VHL-deficient cells, p22(phox)-dependent Nox1 and Nox4 oxidases maintain hypoxia inducible factor-2alpha (HIF-2alpha) protein expression through an Akt-dependent translational pathway. Phosphorylation of tuberin, by Akt, results in its inactivation. Here we show that diphenyleneiodonium chloride, an inhibitor of Nox oxidases, and small-interfering RNA-mediated down-regulation of p22(phox) inhibit Akt-dependent phosphorylation of tuberin and stabilizes tuberin protein levels in VHL-deficient renal carcinoma cells. p22(phox)-mediated inactivation of tuberin is associated with an increase in ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) phosphorylation as well as HIF-2alpha stabilization. Importantly, we find that marked up-regulation of p22(phox) in human renal cell carcinoma correlates with increased tuberin phosphorylation, decreased tuberin protein levels, and increased phosphorylation of 4E-BP1. Our data provide the first evidence that p22(phox)-based Nox oxidases maintain HIF-2alpha protein expression through inactivation of tuberin and downstream activation of ribosomal protein S6 kinase 1/4E-BP1 pathway.

Sudden cardiac death in hemodialysis patients: an in-depth review.

Sudden cardiac death (SCD) is the leading cause of death in hemodialysis patients, accounting for death in up to one-quarter of this population. Unlike in the general population, coronary artery disease and heart failure often are not the underlying pathologic processes for SCD; accordingly, current risk stratification tools are inadequate when assessing these patients. Factors assuming greater importance in hemodialysis patients may include left ventricular hypertrophy, electrolyte shift, and vascular calcification. Knowledge regarding SCD in hemodialysis patients is insufficient, in part reflecting the lack of an agreed-on definition of SCD in this population, although epidemiologic studies suggest the most common times for SCD to occur are toward the end of the long 72-hour weekend interval between dialysis sessions and in the 12 hours immediately after hemodialysis. Accordingly, it is hypothesized that the dialysis procedure itself may have important implications for SCD. Supporting this is recognition that hemodialysis is associated with both ventricular arrhythmias and dynamic electrocardiographic changes. Importantly, echocardiography and electrocardiography may show changes that are modifiable by alterations to dialysis prescription. The most effective preventative strategy in the general population, implanted cardioverter-defibrillator devices, are less effective in the presence of chronic kidney disease and have not been studied adequately in dialysis patients. Last, many dialysis patients experience SCD despite not fulfilling current criteria for implantation, making appropriate allocation of defibrillators uncertain.

Synthesis of substituted N-[3-(3-methoxyphenyl)propyl] amides as highly potent MT(2)-selective melatonin ligands.

A series of substituted N-[3-(3-methoxyphenyl)propyl] amides were synthesized and their binding affinities towards human melatonin MT(1) and MT(2) receptors were evaluated. It was discovered that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT(2) binding affinity and at the same time decreased MT(1) binding affinity.

Rituximab for rescue and maintenance therapy in rapidly progressive life-threatening antineutrophil cytoplasmic autoantibody-associated systemic vasculitis.

Antineutrophil cytoplasmic autoantibody (ANCA)-associated systemic vasculitides (AASV) consists of small-vessel systemic inflammatory disorders which commonly affect the kidneys and without treatment have a poor prognosis. Rituximab is a novel biological agent which is being used experimentally in the management of AASV. This report presents the case of a young woman with rapidly progressive life-threatening AASV. Despite prompt diagnosis and initial treatment with steroids and alkylating agents her condition became life threatening. With addition of rituximab therapy she showed an excellent sustained response. Rituximab appears an effective and safe treatment choice for the induction of remission in severe AASV that is not responding to standard agents, at the initial presentation and for maintenance therapy, without the development of common serious side-effects associated with immunosuppression.

Self-collection: An appropriate alternative during the SARS-CoV-2 pandemic.

OBJECTIVES: To evaluate the reliability of self-collection for SARS-CoV-2 and other respiratory viruses because swab collections for SARS-CoV-2 put health workers at risk of infection and require use of personal protective equipment (PPE). METHODS: In a prospective study, patients from two states in Australia attending dedicated COVID-19 collection clinics were offered the option to first self-collect (SC) nasal and throat swabs (SCNT) prior to health worker collect (HC) using throat and nasal swabs (Site 1) or throat and nasopharyngeal swabs (Site 2). Samples were analysed for SARS-CoV-2 as well as common respiratory viruses. Concordance of results between methods was assessed using Cohen's kappa (κ) and Cycle threshold (Ct) values were recorded for all positive results as a surrogate measure for viral load. RESULTS: Of 236 patients sampled by HC and SC, 25 had SARS-CoV-2 (24 by HC and 25 by SC) and 63 had other respiratory viruses (56 by HC and 58 by SC). SC was highly concordant with HC (κ = 0.890) for all viruses including SARS-CoV-2 and more concordant than HC to positive results by any method (κ = 0.959 vs 0.933). Mean SARS-CoV-2 E-gene and N-gene, rhinovirus and parainfluenza Ct values did not differ between HC and SCNT. CONCLUSIONS: Self-collection of nasal and throat swabs offers a reliable alternative to health worker collection for the diagnosis of SARS-CoV-2 and other respiratory viruses and provides patients with easier access to testing, reduces exposure of the community and health workers to those being tested and reduces requirement for PPE.

Reporter gene assays.

Reporter gene assays are versatile and sensitive methods of assaying numerous targets in high-throughput drug-screening programs. A variety of reporter genes allow users a choice of signal that can be tailored to the required sensitivity, the available detection apparatus, the cellular system employed, and the required compatibility with multiplexed assays. Promoters used to drive reporter gene expression can be activated either by a broad range of biochemical pathways or by the selective activation of individual targets. In this chapter, we will introduce some of the considerations behind the choice of reporter gene assays and describe the methods that we have used to establish 96-well format luciferase and aequorin assays for the screening of ligands for G protein-coupled receptors.

Case Reports: Late Emergence of Cutaneous Leishmaniasis in an Immunocompromised Patient in a Non-Endemic Setting.

We report a case of reactivation of cutaneous leishmaniasis caused by Leishmania major in a 40-year-old man from Afghanistan, in the context of immune suppression. His last potential exposure was 7 years before the clinical presentation. To our knowledge, this is the most temporally distant reactivation of cutaneous leishmaniasis reported in a non-endemic country.

G protein-coupled receptor-induced Akt activity in cellular proliferation and apoptosis.

Akt (also known as protein kinase B) plays an integral role in many intracellular signaling pathways activated by a diverse array of extracellular signals that target several different classes of membrane-bound receptors. Akt plays a particularly prominent part in signaling networks that result in the modulation of cellular proliferation, apoptosis and survival. Thus, the overexpression of Akt subtypes has been measured in a number of cancer types, and dominant-negative forms of Akt can trigger apoptosis and reduce the survival of cancer cells. G protein-coupled receptors act as cell-surface detectors for a diverse spectrum of biological signals and are able to activate or inhibit Akt via several direct and indirect means. In this review, we shall document how G protein-coupled receptors are able to control Akt activity and examine the resulting biochemical and physiological changes, with particular emphasis on cellular proliferation, apoptosis and survival.

Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis.

BACKGROUND: We investigated sera from elderly subjects with and without age-related macular degeneration (AMD) for presence of autoantibodies (AAbs) against human macular antigens and characterized their identity. METHODS: Sera were collected from participants in the Age-Related Maculopathy Ancillary (ARMA) Study, a cross-sectional investigation ancillary to the Health ABC Study, enriched with participants from the general population. The resulting sample (mean age: 79.2±3.9 years old) included subjects with early to advanced AMD (n = 131) and controls (n = 231). Sera were tested by Western blots for immunoreactive bands against human donor macular tissue homogenates. Immunoreactive bands were identified and graded, and odds ratios (OR) calculated. Based on these findings, sera were immunoprecipitated, and subjected to 2D gel electrophoresis (GE). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities. RESULTS: In AMD, 11 immunoreactive bands were significantly more frequent and 13 were significantly stronger than in controls. Nine of the more frequent bands also showed stronger reactivity. OR estimates ranged between 4.06 and 1.93, and all clearly excluded the null value. Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin. ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls. CONCLUSIONS: Consistent with other evidence supporting the role of inflammation and the immune system in AMD pathogenesis, AAbs were identified in AMD sera, including early-stage disease. Identified targets may be mechanistically linked to AMD pathogenesis because the identified proteins are implicated in autophagy, immunomodulation, and protection from oxidative stress and apoptosis. In particular, a role in autophagy activation is shared by all five autoantigens, raising the possibility that the detected AAbs may play a role in AMD via autophagy compromise and downstream activation of the inflammasome. Thus, we propose that the detected AAbs provide further insight into AMD pathogenesis and have the potential to contribute to disease biogenesis and progression.

BML-190 and AM251 act as inverse agonists at the human cannabinoid CB2 receptor: signalling via cAMP and inositol phosphates.

The aminoalkylindole BML-190 and diarylpyrazole AM251 ligands have previously been shown to bind to cannabinoid CB(2) and CB(1) receptors, respectively. In HEK-293 cells stably expressing the human CB(2) receptor, BML-190 and AM251 potentiated the forskolin-stimulated accumulation of cAMP. Moreover, the CB(2) receptor can interact productively with 16z44, a promiscuous G alpha(16/z) chimera. BML-190 and AM251 reduce the basal levels of inositol phosphate production in cells expressing the CB(2) receptor and 16z44. These results demonstrate that BML-190 and AM251 act as inverse agonists at the human CB(2) receptor acting via G alpha(i/o) and G alpha(q) family-coupled pathways.