Comparison of adherence measures using claims data in the South African private health sector

Background. Medication adherence measurement is becoming increasingly important. Biological assays and markers, directly observed therapy, self-reports, pill counts and surveys have been successfully used to assess adherence under various circumstances, but may be limited by cost, ethical concerns and self-reported bias. Administrative claims data, in addition to offering a solution to these limitations, provide access to large study populations under real clinical practice situations, and in a timely and effective manner. With the wide range of adherence measures determined from claims data available – some of which have been found to be mathematically equivalent – researchers are often faced with the decision of choosing which is appropriate. An assessment of the various measures is therefore important for better understanding and to facilitate future adherence studies using administrative data.Objectives. To compare different adherence measures using data from a medicines claims database in South Africa (SA), employing montelukast for the purpose of illustration.Methods. This retrospective, cross-sectional research used data from 1 January 2006 to 31 December 2015 from a privately owned pharmaceutical benefits management (PBM) company in SA. Claims for montelukast were identified and adherence was determined using the continuous multiple-interval measure of oversupply (CMOS), compliance ratio (CR), modified medication possession ratio (MPRm), refill compliance rate (RCR), continuous single-interval measure of medication acquisition (CSA) and proportion of days covered (PDC) capped at 1. The measures were compared with the medication possession ratio (MPR) as the reference.Results. The MPR, CMOS and CR were equivalent, each yielding an adherence value of 86%. The MPRm, RCR and average CSA yielded higher adherence values of 96.9%, 117.2% and 129.0%, respectively, whereas the PDC produced a lower adherence value of 76.0%. The measures that used the entire study period as the denominator produced consistent results compared with the measures that used the difference between claims dates as denominator.Conclusions. The MPR is considered the most widely used metric to measure adherence using administrative data, but it may not always be applicable owing to the type of data available. Adherence computed using the CR, CMOS and PDC capped was found to be comparable to the MPR, and they may therefore be used as alternatives.

Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

<p>Abstract</p> <p>Background</p> <p>The role of protein kinase C (PKC) in regulating the activity of phospholipase C (PLC) in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM), was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM) and staurosporine (400 nM).</p> <p>Methods</p> <p>Alterations in cytosolic Ca²⁺, Ca²⁺influx, inositol triphosphate (IP₃), and leukotriene B₄production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively.</p> <p>Results</p> <p>Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca²⁺followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP₃production with associated enhanced Ca²⁺release from storage vesicles, prolongation of the peak cytosolic Ca²⁺transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB₄. The alterations in Ca²⁺fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca²⁺-resequestering endomembrane ATPase.</p> <p>Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP₃production and down-regulation of Ca²⁺mediated pro-inflammatory responses of PAF-activated neutrophils.</p> <p>Conclusion</p> <p>Although generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.</p

The role of pneumolysin in mediating lung damage in a lethal pneumococcal pneumonia murine model

BACKGROUND: Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia. METHODS: To examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice. RESULTS: In bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues. CONCLUSION: The data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival

Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B

<p>Abstract</p> <p>Background</p> <p>Nontypeable <it>Haemophilus influenzae </it>(NTHi) is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) via activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B in pulmonary alveolar epithelial cells.</p> <p>Methods</p> <p>Human alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA), and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The roles of Toll-like receptor (TLR) 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4 <it>in vitro </it>and in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out mice <it>in vivo</it>. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors.</p> <p>Results</p> <p>NTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cell <it>in vitro </it>and in the lungs of mice <it>in vivo</it>. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B. Moreover, the expressions of COX-2 and PGE2 were significantly inhibited by specific inhibitors of p38 MAPK and NF-kappa B. However, NTHi-induced DNA binding activity of NF-kappa B was not affected by the inhibition of p38 MAPK.</p> <p>Conclusion</p> <p>NTHi induces COX-2 and PGE2 expression in a p38 MAPK and NF-kappa B-dependent manner through TLR2 in lung epithelial cells <it>in vitro </it>and lung tissues <it>in vivo</it>. The full understanding of the role of endogenous anti-inflammatory PGE2 and its regulation will bring new insight to the resolution of inflammation in pulmonary bacterial infections.</p

Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system

Differential activation of inflammatory pathways in A549 type II pneumocytes by Streptococcus pneumoniae strains with different adherence properties

BACKGROUND: Adherence of Streptococcus pneumoniae bacteria to lung cells is a first step in the progression from asymptomatic carriage to pneumonia. Adherence abilities vary widely among S. pneumoniae patient isolates. In this study, the binding properties of S. pneumoniae isolates and the effects of binding on activation of the Nuclear Factor-Kappa-B (NFκB) pathway and cytokine secretion by type II pneumocytes were measured. METHODS: Mechanisms of high- and low-binding S. pneumoniae adherence to A549 cells were investigated by blocking putative receptors on bacteria and host cells with antibody and by eluting choline-binding proteins off of bacterial surfaces. NFκB activation was measured by western blot and immunocytochemistry and cytokine secretion was detected by a protein array. RESULTS: This study shows that S. pneumoniae isolates from pneumonia patients (n = 298) can vary by as much as 1000-fold in their ability to bind to human lung epithelial cells. This difference resulted in differential activation of the NFκB pathway. High-, but not low-binding S. pneumoniae used Choline-binding protein A (CbpA) to bind to complement component C3 on epithelial cell surfaces. Interleukin-8 (IL-8) was the only cytokine secreted by cells treated with either low- or high-binding S. pneumoniae. CONCLUSION: These results indicate that S. pneumoniae clinical isolates are not homogeneous in their interaction with host epithelial cells. The differential activation of host cells by high- and low-binding S. pneumoniae strains could have implications for the treatment of pneumococcal pneumonia and for vaccine development

ε/ζ systems: their role in resistance, virulence, and their potential for antibiotic development

Cell death in bacteria can be triggered by activation of self-inflicted molecular mechanisms. Pathogenic bacteria often make use of suicide mechanisms in which the death of individual cells benefits survival of the population. Important elements for programmed cell death in bacteria are proteinaceous toxin–antitoxin systems. While the toxin generally resides dormant in the bacterial cytosol in complex with its antitoxin, conditions such as impaired de novo synthesis of the antitoxin or nutritional stress lead to antitoxin degradation and toxin activation. A widespread toxin–antitoxin family consists of the ε/ζ systems, which are distributed over plasmids and chromosomes of various pathogenic bacteria. In its inactive state, the bacteriotoxic ζ toxin protein is inhibited by its cognate antitoxin ε. Upon degradation of ε, the ζ toxin is released allowing this enzyme to poison bacterial cell wall synthesis, which eventually triggers autolysis. ε/ζ systems ensure stable plasmid inheritance by inducing death in plasmid-deprived offspring cells. In contrast, chromosomally encoded ε/ζ systems were reported to contribute to virulence of pathogenic bacteria, possibly by inducing autolysis in individual cells under stressful conditions. The capability of toxin–antitoxin systems to kill bacteria has made them potential targets for new therapeutic compounds. Toxin activation could be hijacked to induce suicide of bacteria. Likewise, the unique mechanism of ζ toxins could serve as template for new drugs. Contrarily, inhibition of virulence-associated ζ toxins might attenuate infections. Here we provide an overview of ε/ζ toxin–antitoxin family and its potential role in the development of new therapeutic approaches in microbial defense

Pneumolysin induces release of matrix metalloproteinase-8 and -9 from human neutrophils

The research question addressed in the current study was: does the pneumococcal pore-forming toxin, pneumolysin, mobilise matrix metalloproteinase (MMP) -8 and -9 from isolated human blood neutrophils at sublytic concentrations of 5, 10 and 20 ng.mL(-1)? MMPs were measured in the supernatants of unstimulated neutrophils and of cells exposed to pneumolysin and the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (f-MLP; 0.1 mu M), individually and in combination, using ELISA procedures, and alterations in cytosolic Ca2+ concentrations were monitored using a fura-2 acetoxymethyl ester (fura-2/AM)-based spectrofluorimetric method. Treatment of neutrophils with pneumolysin alone caused dose-related release of both MMPs, whereas f-MLP caused modest increases; the combination of both activators was, however, most effective. Pneumolysin/f-MLP-activated release of the MMPs from the cells was paralleled by increases in cytosolic Ca2+. Exposure of human neutrophils to pneumolysin is accompanied by mobilisation of MMPs, which is potentiated by f-MLP. If operative in vivo, pneumolysin-mediated release of MMPs from neutrophils and other cell types may contribute to the pathogenesis of severe pneumococcal disease

Hyaluronidase augments pneumolysin-mediated injury to human ciliated epithelium

SummaryObjectivesThe main objective of this study was to investigate the effects of pneumococcal hyaluronidase (0.1–10μg/ml), alone and in combination with pneumolysin (50 and 100ng/ml), on human ciliated epithelium.MethodsCiliary beat frequency (CBF) and structural integrity of human ciliated respiratory epithelium in vitro were studied using a phototransistor technique and a visual scoring index, respectively.ResultsHyaluronidase per se did not affect either CBF or the structural integrity of the epithelium. However, preincubation of the epithelial strips with hyaluronidase (10μg/ml) for 30min at 37°C significantly potentiated pneumolysin-mediated ciliary slowing and epithelial damage. Hyaluronan, a substrate of hyaluronidase, had no effects on the ciliated respiratory epithelium in concentrations up to 100μg/ml and did not antagonize the injurious effects of pneumolysin on the epithelium. However, preincubation of the epithelial strips with hyaluronan (100μg/ml) was associated with attenuation of the ciliary slowing and epithelial damage induced by incubation of the strips with hyaluronidase (10μg/ml) for 30min at 37°C followed by addition of pneumolysin (50ng/ml).ConclusionsAlthough having no direct effects alone, hyaluronidase may contribute to pneumolysin-mediated damage and dysfunction to respiratory epithelium, thereby favoring colonization and subsequently extra-pulmonary dissemination of the pneumococcus