Comparison of the effects of electronic cigarette vapours and tobacco smoke extracts on human neutrophils in vitro.

Background: Electronic cigarettes (ECs) are electronic aerosol delivery systems composed of nicotine and various chemicals, which are widely used to facilitate smoking cessation. Although ECs are considered safer than cigarettes, they do, however, contain chemical toxicants, some of which may interact with cells of the host's innate immune system of which neutrophils constitute a key component. Methods: The current study was designed to compare the effects of aqueous EC aerosol extracts (ECEs; with or without nicotine) with those of cigarette smoke extract (CSE) on neutrophil and platelet reactivity in vitro. Neutrophil reactivity is characterised by the generation of reactive oxygen species (ROS), degranulation (elastase release) and the release of extracellular DNA (neutrophil extracellular trap (NET) formation: NETosis), which were measured using chemiluminescence, spectrophotometric and microscopic procedures, respectively. Platelet reactivity was measured according to the magnitude of upregulated expression of the adhesion molecule CD62P on activated cells using a flow cytometric procedure. Results: Exposure of neutrophils to either ECEs or CSE caused a significant inhibition of ROS generation and elastase release by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 µM)-activated neutrophils. Pre-treatment of neutrophils with CSE also resulted in a marked attenuation of phorbol 12-myristate 13-acetate (6.25 nM)-mediated release of extracellular DNA, which was unaffected by the ECEs. Similarly, CSE, but not the ECEs, inhibited the expression of CD62P by platelets activated with ADP (100 µM). Conclusions: These observations suggest that ECE aerosols may inhibit some of the immuno-protective activities of neutrophils such as ROS production and elastase release by activated cells, the effect of which was not enhanced by inclusion of nicotine. The inhibitory effects of CSE were significantly more pronounced than those of ECEs, especially so for suppression of NET formation and platelet activation. If operative in vivo, these harmful immunosuppressive effects of ECEs may compromise intrinsic pulmonary antimicrobial defence mechanisms, albeit less so than cigarette smoke.

Pro-Inflammatory Interactions of Dolutegravir with Human Neutrophils in an In Vitro Study.

There is increasing awareness of an association between the uptake of the HIV integrase inhibitor, dolutegravir, in first-line antiretroviral regimens with unusual weight gain and development of the metabolic syndrome, particularly in African women. Although seemingly unexplored, the development of systemic inflammation linked to the putative pro-inflammatory activity of dolutegravir represents a plausible pathophysiological mechanism of this unusual weight gain. This possibility was explored in the current study undertaken to investigate the effects of dolutegravir (2.5−20 µg/mL) on several pro-inflammatory activities of neutrophils isolated from the blood of healthy, adult humans. These activities included the generation of reactive oxygen species (ROS), degranulation (elastase release) and alterations in the concentrations of cytosolic Ca2+ using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of neutrophils to dolutegravir alone resulted in the abrupt, dose-related, and significant (p < 0.0039−p < 0.0022) generation of ROS that was attenuated by the inclusion of the Ca2+-chelating agent, EGTA, or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI), phospholipase C (U733122), myeloperoxidase (sodium azide) and phosphoinositol-3-kinase (wortmannin). In addition, exposure to dolutegravir augmented the release of elastase by stimulus-activated neutrophils. These pro-inflammatory effects of dolutegravir on neutrophils were associated with significant, rapid, and sustained increases in the concentrations of cytosolic Ca2+ that appeared to originate from the extracellular compartment, seemingly consistent with an ionophore-like property of dolutegravir. These findings are preliminary and necessitate verification in the clinical setting of HIV infection. Nevertheless, given the complex link between inflammation and obesity, these pro-inflammatory interactions of dolutegravir with neutrophils may contribute to unexplained weight gain, possibly via the development of insulin resistance.

Dolutegravir potentiates platelet activation by a calcium-dependent, ionophore-like mechanism.

Dolutegravir is a highly potent HIV integrase strand transfer inhibitor that is recommended for first-line anti-retroviral treatment in all major treatment guidelines. A recent study has shown that people taking this class of anti-retroviral treatment have a substantially higher risk of early-onset cardiovascular disease, a condition shown previously to be associated with increased platelet reactivity. To date, few studies have explored the effects of dolutegravir on platelet activation. Accordingly, the current study was undertaken with the primary objective of investigating the effects of dolutegravir on the reactivity of human platelets in vitro. Platelet-rich plasma, isolated platelets, or buffy coat cell suspensions prepared from the blood of healthy adults were treated with dolutegravir (2.5-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin, or a thromboxane A2 receptor agonist U46619. Expression of platelet CD62P (P-selectin), formation of heterotypic neutrophil:platelet aggregates, and calcium (Ca2+) fluxes were measured using flow cytometry and fluorescence spectrometry, respectively. Dolutegravir caused dose-related potentiation of ADP-, thrombin- and U46619-activated expression of CD62P by platelets, as well as a significant increases in formation of neutrophil:platelet aggregates. These effects were paralleled by a spontaneous, receptor-independent elevation in cytosolic Ca2+ that appears to underpin the mechanism by which the antiretroviral agent augments the responsiveness of these cells to ADP, thrombin and U46619. The most likely mechanism of dolutegravir-mediated increases in platelet cytosolic Ca2+ relates to a combination of lipophilicity and divalent/trivalent metal-binding and/or chelating properties of the anti-retroviral agent. These properties are likely to confer ionophore-type activities on dolutegravir that would promote movement of Ca2+ across the plasma membrane, delivering the cation to the cytosol where it would augment Ca2+-dependent intracellular signaling mechanisms. These effects of dolutegravir may lead to hyper-activation of platelets which, if operative in vivo, may contribute to an increased risk for cardiometabolic co-morbidities.

Ex vivo platelet morphology assessment of chronic myeloid leukemia patients before and after Imatinib treatment.

Chronic myeloid leukemia (CML) is a myeloproliferative disease and the first line treatment is through the administration of Imatinib, a first generation tyrosine kinase inhibitor. Thrombocytosis and bleeding irregularities are common in CML, however, the morphological variations in CML patients' platelets are not well documented. In this study, ex vivo platelet morphology of control participants, as well as CML patients were assessed before and after Imatinib treatment. The topographical and structural morphology of platelets were determined via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Qualitative data of SEM and TEM revealed that CML patient's platelets were prone to aggregation and coagulation at time of diagnosis; the samples that were not aggregated at time of diagnosis showed typical discoid shaped platelets, which was comparable to control participants' platelets. TEM results of CML patients' platelets at diagnosis showed that internal granular constituents including dense bodies were decreased in comparison to control participants. In all CML patients, platelets appeared activated after 6 months of treatment with Imatinib with membrane structure abnormalities and constituent variations. Research to date has primarily focused on the effects of CML on leukocyte populations, however, the results of the current study implicate the impact of CML pathogenesis on platelets, seemingly as a result of alterations in normal hematopoiesis. In addition, the impact of Imatinib treatment on platelet morphology was also established, indicating an increase in platelet activation. Recognizing and understanding the impact of CML disease progression on platelets is of importance to aid improved patient treatment. RESEARCH HIGHLIGHTS: In the study, results from SEM and TEM indicated that CML patient's platelets were prone to aggregation at time of diagnosis, and activation after Imatnib treatment. Platelet samples that did not aggregate had decreased internal granular constituents.

Pulmonary Toxicities Associated With the Use of Immune Checkpoint Inhibitors: An Update From the Immuno-Oncology Subgroup of the Neutropenia, Infection & Myelosuppression Study Group of the Multinational Association for Supportive Care in Cancer.

The development of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, with agents such as nivolumab, pembrolizumab, and cemiplimab targeting programmed cell death protein-1 (PD-1) and durvalumab, avelumab, and atezolizumab targeting PD-ligand 1 (PD-L1). Ipilimumab targets cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). These inhibitors have shown remarkable efficacy in melanoma, lung cancer, urothelial cancer, and a variety of solid tumors, either as single agents or in combination with other anticancer modalities. Additional indications are continuing to evolve. Checkpoint inhibitors are associated with less toxicity when compared to chemotherapy. These agents enhance the antitumor immune response and produce side- effects known as immune-related adverse events (irAEs). Although the incidence of immune checkpoint inhibitor pneumonitis (ICI-Pneumonitis) is relatively low, this complication is likely to cause the delay or cessation of immunotherapy and, in severe cases, may be associated with treatment-related mortality. The primary mechanism of ICI-Pneumonitis remains unclear, but it is believed to be associated with the immune dysregulation caused by ICIs. The development of irAEs may be related to increased T cell activity against cross-antigens expressed in tumor and normal tissues. Treatment with ICIs is associated with an increased number of activated alveolar T cells and reduced activity of the anti-inflammatory Treg phenotype, leading to dysregulation of T cell activity. This review discusses the pathogenesis of alveolar pneumonitis and the incidence, diagnosis, and clinical management of pulmonary toxicity, as well as the pulmonary complications of ICIs, either as monotherapy or in combination with other anticancer modalities, such as thoracic radiotherapy.

The COVID-19 Treatment Landscape: A South African Perspective on a Race Against Time.

The pandemic caused by SARS-CoV-2 has infected more than 94 million people worldwide (as of 17 January 2020). Severe disease is believed to be secondary to the cytokine release syndrome (CRS or "cytokine storm") which causes local tissue damage as well as multi-organ dysfunction and thrombotic complications. Due to the high mortality rates in patients receiving invasive ventilation, practice has changed from "early-intubation" for acute respiratory distress syndrome (ARDS) to a trial of non-invasive ventilation (NIV) or high flow nasal cannula (HFNC) oxygen. Reports indicating the benefit of NIV and HFNC have been encouraging and have led to more than 20,000 such devices being manufactured and ready for roll-out in South Africa (SA) as of July 2020. The need to identify drugs with clear clinical benefits has led to an array of clinical trials, most of which are repurposing drugs for COVID-19. The treatment landscape reflects the need to target both the virus and its effects such as the CRS and thrombotic complications. Conflicting results have the potential to confuse the implementation of coordinated treatment strategies and guidelines. The purpose of this review is to address pertinent areas in the current literature on the available medical treatment options for COVID-19. Remdesivir, tocilizumab, and dexamethasone are some of the treatment options that have shown the most promise, but further randomized trials are required to particularly address timing and dosages to confidently create standardized protocols. For the SA population, two healthcare sectors exist. In the private sector, patients with medical insurance may have greater access to a wider range of treatment options than those in the public sector. The latter serves >80% of the population, and resource constraints require the identification of drugs with the most cost-effective use for the greatest number of affected patients.

Apoptotic profiling of chronic myeloid leukaemia patients' platelets ex vivo before and after treatment with Imatinib.

Chronic myeloid leukaemia (CML) is a malignancy of the haematopoietic stem cells. The first line of treatment for CML, especially in developing countries, remains the first-generation tyrosine kinase inhibitor, Imatinib. Patients with CML are frequently diagnosed with platelet abnormalities. However, the specific mechanism of platelet abnormalities in CML remains unclear and poorly understood. The aim of this study was therefore to determine the apoptotic profiles of CML patients ex vivo on platelets before and after treatment with Imatinib. Blood samples of healthy volunteers and CML patients at diagnosis and after 6 months treatment with Imatinib were collected. Platelet counts, viability and activation were determined. Results showed that CML patients' platelet counts were elevated upon diagnosis and these levels statistically significantly decreased after 6 months of treatment. Platelet activation was significantly increased after 6 months of treatment compared to levels at diagnosis (P-value < .05). Similarly, platelet apoptosis was also increased after 6 months of treatment. Abnormalities in platelet functioning found in this study may partly be due to clonal proliferation of haematopoietic cells in CML patients, specifically of megakaryocyte precursors as well as the inhibition of platelet tyrosine kinase's and the inhibition of platelet-derived growth factor.

ADP-Mediated Upregulation of Expression of CD62P on Human Platelets Is Critically Dependent on Co-Activation of P2Y1 and P2Y12 Receptors.

This study probed the differential utilization of P2Y1 and P2Y12 receptors in mobilizing CD62P (P-selectin) from intracellular granules following activation of human platelets with adenosine 5'-diphosphate (ADP, 100 µmol·L-1) Platelet-rich plasma (PRP) was prepared from the blood of adult humans. CD62P was measured by flow cytometry following activation of PRP with ADP in the absence and presence of the selective antagonists of P2Y1 and P2Y12 receptors, MRS2500 and PSB0739 (both 0.155-10 µmol·L-1), respectively. Effects of the test agents on ADP-activated, CD62P-dependent formation of neutrophil:platelet (NP) aggregates were also measured by flow cytometry, while phosphatidylinositol 3-kinase (PI3K) activity was measured according to Akt1 phosphorylation in platelet lysates. Treatment with MRS2500 or PSB0739 at 10 µmol·L-1 almost completely attenuated (94.6% and 86% inhibition, respectively) ADP-activated expression of CD62P and also inhibited NP aggregate formation. To probe the mechanisms involved in P2Y1/P2Y12 receptor-mediated expression of CD62P, PRP was pre-treated with U73122 (phospholipase C (PLC) inhibitor), 2-aminoethoxy-diphenyl borate (2-APB, inositol triphosphate receptor antagonist), calmidazolium chloride (calmodulin inhibitor), or wortmannin (PI3K inhibitor). U73122, 2-APB, and wortmannin caused almost complete inhibition of ADP-activated expression of CD62P, while calmidazolium chloride caused statistically significant, partial inhibition. PSB0739, but not MRS2500, caused potent inhibition of PI3K-mediated phosphorylation of Akt1. Optimal mobilization of CD62P by ADP-stimulated platelets is critically dependent on the co-activation of platelet P2Y1 and P2Y12 receptors. P2Y12 receptor activation is the key event in activation of PI3K, while activation of the P2Y1 receptor appears to create a high cytosolic Ca2+ environment conducive to optimum PI3K activity.

Submission for Special Issue: The Role of Platelet Activation in the Pathophysiology of HIV, Tuberculosis, and Pneumococcal Disease. Bedaquiline Suppresses ADP-Mediated Activation of Human Platelets In Vitro via Interference With Phosphatidylinositol 3-Kinase.

Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 µg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.

Tobacco-Derived Lipopolysaccharide, Not Microbial Translocation, as a Potential Contributor to the Pathogenesis of Rheumatoid Arthritis.

Microbial lipopolysaccharides (LPS) have been implicated in the pathogenesis of rheumatoid arthritis (RA), possibly driving a systemic inflammatory response that may trigger the development and/or exacerbation of the disease. To explore the existence of this mechanism in African RA patients, we have measured systemic levels of LPS and its surrogate, LPS-binding protein (LBP), as well as those of intestinal fatty acid-binding protein (I-FABP), pulmonary surfactant protein D (SP-D), and cotinine in serum to identify possible origins of LPS, as well as associations of these biomarkers with rheumatoid factor (RF) and anticitrullinated peptide (aCCP) autoantibodies and the DAS 28-3 clinical disease severity score. A cohort of 40 disease-modifying antirheumatic drug-naïve, black South African RA patients rated by compound disease scores and 20 healthy subjects and 10 patients with chronic obstructive pulmonary disease (COPD) as controls were included in this study. Levels of the various biomarkers and autoantibodies were measured using a combination of ELISA and immunofluorimetric and immunoturbidometric procedures. LPS levels were lowest in the RA group compared to the healthy controls (p = 0.026) and COPD patients (p = 0.017), while LBP levels were also significantly lower in RA compared to the healthy individuals (p = 0.036). Levels of I-FABP and SP-D were comparable between all three groups. Categorisation of RA patients according to tobacco usage revealed the following significant positive correlations: LBP with C-reactive protein (p = 0.0137); a trend (p = 0.073) towards an association of LBP with the DAS 28-3 disease severity score; RF-IgG antibodies with both LPS and LBP (p = 0.033 and p = 0.041, respectively); aCCP-IgG antibodies with LPS (p = 0.044); and aCCP-IgG with RF-IgM autoantibodies (p = 0.0016). The findings of this study, several of them novel, imply that tobacco products, as opposed to microbial translocation, represent a potential source of LPS in this study cohort of RA patients, again underscoring the risks posed by tobacco usage for the development and severity of RA.

Interleukin-10 and interleukin-1 receptor antagonist distinguish between patients with sepsis and the systemic inflammatory response syndrome (SIRS).

The current study evaluated the potential of clinical parameters and circulating biomarkers to distinguish sepsis from SIRS in patients admitted with systemic inflammation. Clinical parameters, leukocyte counts and platelets were measured on admission. Circulating C-reactive protein (CRP), procalcitonin (PCT) and cytokine concentrations were quantified using laser immunonephelometry, immunoluminescence and a Bio-Plex suspension bead array system respectively. Blood, sputum, urine, peritoneal and cerebrospinal fluid were sent for microscopy and culture. Based on clinical information and the results of microbiological testing, 62 patients were classified retrospectively into 2 groups, those with sepsis (n = 37) or SIRS (n = 25). Mean body temperature was higher and blood pressure lower in the sepsis patients. Circulating concentrations of CRP, PCT, interleukin (IL)-10 and IL-1 receptor antagonist (IL-1Ra) were significantly higher in patients with sepsis, with IL-10 identified as the best biomarker in differentiating sepsis from SIRS. The biomarkers that best predicted overall mortality were platelet counts >PCT ≥ CRP > IL-6 > IL-1Ra. These findings demonstrate that patients with sepsis have significantly increased levels of the immunosuppressive/anti-inflammatory cytokines, IL-1Ra and IL-10, compared to those with SIRS, consistent with a more intense counteracting anti-inflammatory response, while a biomarker profile including platelets, PCT, CRP, IL-6 and IL-1Ra may be useful to predict mortality.

Electronic cigarettes: where to from here?

Although the usage of electronic (e)-cigarettes (EC) and similar devices has gained in popularity as an apparent smoking cessation strategy, serious concerns are emerging in relation to both the efficacy of this strategy, as well as the inappropriate use of these devices. While the comparative safety of e-cigarettes is based on the reasonable contention that the levels of inhaled toxicants present in the aerosols generated by these devices are considerably lower than those present in tobacco smoke, the perception that they are indeed relatively risk-free is being challenged on several fronts. Notwithstanding lack of convincing evidence of efficacy as a superior smoking cessation strategy, foremost among emerging concerns is the increasing use of electronic nicotine-delivery devices by young never-smokers. Other concerns include increasing levels of sophistication in the design and capacity of these devices in relation to nicotine content and delivery, the potential threat of manipulation of the contents of e-liquids, as well as other additives such as illicit drugs and other potentially toxic agents that can be vaporised. These issues, together with the potential risks to respiratory health, specifically "e-cigarette or vaping product use-associated lung injury" represent the major thrusts of this review.

Clofazimine, but Not Isoniazid or Rifampicin, Augments Platelet Activation in vitro.

Although the inclusion of the cationic amphiphilic, anti-mycobacterial agent, clofazimine, in the chemotherapeutic regimens of patients with multidrug-resistant tuberculosis (TB) has contributed to improved outcomes, concerns remain about the cardiotoxic potential of this agent. Accordingly, the current study was undertaken with the primary objective of investigating the effects of clofazimine, on the reactivity of human platelets in vitro, a seemingly unexplored, mechanism of cardiotoxicity. Platelet-rich plasma (PRP) prepared from the blood of healthy, adult humans was treated with clofazimine (0.625-10 mg/L), or the primary anti-TB agents, isoniazid and rifampicin (at final concentrations of 5 and 10 mg/L), followed by addition of either adenosine 5'-diphosphate (ADP) or thrombin and measurement of platelet activation according to the magnitude of expression of CD62P (P-selectin), as well as the CD62P-mediated formation of heterotypic neutrophil:platelet (NP) aggregates, using flow cytometry. Clofazimine, but neither isoniazid nor rifampicin, caused dose-related potentiation of both ADP- and thrombin-activated expression of CD62P by platelets, achieving statistical significance at threshold concentrations of 0.625 and 2.5 mg/L, respectively, as well as significant formation of N:P aggregates. These stimulatory effects of clofazimine on platelet activation were partly attenuated by pre-treatment of PRP with the membrane-stabilizing agent, α-tocopherol, possibly consistent with a membrane-disruptive mechanism. In conclusion, clofazimine, at concentrations within the therapeutic range, augments platelet activation in vitro, probably by a mechanism linked to membrane destabilization. If operative in vivo, these pro-thrombotic activities of clofazimine may predispose for development of microvascular occlusion, exacerbating an already existing high risk for development of TB-associated cardiovascular disease.

The Effects of Dabigatran and Rivaroxaban on Markers of Polymorphonuclear Leukocyte Activation.

Dabigatran is an oral direct thrombin inhibitor, and rivaroxaban, a factor Xa inhibitor. Dabigatran has been implicated in the etiology of acute coronary syndromes and as these occur following inflammatory changes in the endothelium, we investigated the inflammatory potential of these agents in vitro. In order to do so, polymorphonuclear leukocytes (PMNL) were isolated from heparinized venous blood from non-smoking, healthy adults and exposed to dabigatran or rivaroxaban (0.5⁻10 µM). Generation of reactive oxygen species (ROS), elastase release, cytosolic Ca2+ fluxes, neutrophil extracellular trap (NET) formation and cell viability were measured using chemiluminescence, spectrophotometric and flow cytometric procedures respectively. However, with the exception of modest inhibitory effects on elastase release, neither agent at concentrations of up to 10 µM affected these markers of PMNL activation. Although no pro-inflammatory effects of dabigatran nor any difference between the two test agents were detected in vitro, the existence of a pro-inflammatory mechanism involving the generation of thrombin during dabigatran therapy cannot be fully excluded.

Aortic Arch Baroreceptor Stimulation in an Experimental Goat Model: A Novel Method to Lower Blood Pressure.

The effect of aortic baroreceptor stimulation on blood pressure manipulation was assessed using the goat species Capra aegagrus hircus. The aim of this study was to manipulate blood pressure with future intention to treat high blood pressure in humans. The ages of the animals ranged from 6 months to 2 years. A standard anesthesia protocol was used. A lateral thoracotomy was performed to gain access to the aortic arch. Data was collected with the Vigileo system. Pre stimulation blood pressure was compared with maximum post stimulation blood pressure values. Results were analyzed with the Wilcoxon signed rank test. In the study 38 animals were enrolled. Baroreceptor stimulation was performed for each animal using 3 different electrodes each of which emits an electrical impulse. In the pilot phase of the study, the median baseline blood pressure prior to stimulation of the baroreceptors was 110.8 mmHg. After stimulation the median blood pressure decreased to 88 mmHg. The average decrease in blood pressure was 22.8 mmHg. This decrease of blood pressure after stimulation of the baroreceptors is statistically significant (p < 0.0001) and the proof of concept was shown. During the extended phase all three probes had a significant effect on blood pressure lowering (p < 0.0001). The study confirmed that aortic baroreceptor stimulation has an effect on blood pressure lowering. This is a novel field of blood pressure manipulation. The hemodynamic effects of long-term aortic baroreceptor stimulation are unknown. Further investigations need to be done to determine whether a similar effect can be induced in different species such as primates and humans.

Cigarette smoke condensate attenuates phorbol ester-mediated neutrophil extracellular trap formation.

BACKGROUND: Neutrophil extracellular traps (NETs) constitute a network of chromatin fibres containing histone and antimicrobial peptides that are released by activated neutrophils. NETs protect the host against infection by trapping and facilitating phagocytosis of potentially harmful pathogens. OBJECTIVES: The aim of the current study was to investigate the effects of cigarette smoke condensate (CSC) on phorbol-ester (PMA)-mediated NETosis in vitro. METHODS: Isolated human blood neutrophils were exposed to PMA (6.25 ng/ml) in the presence or absence of CSC (40-80 µg/ml) for 90 min at 37oC. NET formation was measured using a spectrofluorimetric procedure to detect extracellular DNA and fluorescence microscopy was used to visualize nets. Oxygen consumption by PMA-activated neutrophils was measured using an oxygen sensitive electrode. RESULTS: Activation of neutrophils with PMA was associated with induction of NETosis that was significantly attenuated in the presence of CSC (40 and 80 µg/ml), with mean fluorescence intensities of 65% and 66% of that observed with untreated cells, respectively, and confirmed by fluorescence microscopy. The rate and magnitude of oxygen consumption by activated neutrophils pre-treated with CSC (80 µg/ml) was significantly less than that observed with untreated cells (73% of the control system), indicative of decreased production of reactive oxidants in the presence of CSC. CONCLUSION: The inhibition of NETosis observed in the presence of CSC correlated with attenuation of oxygen consumption by PMA-activated neutrophils suggesting a mechanistic relationship between these events. If operative in vivo, smoking-related attenuation of NETosis may impair host immune responses and increase the risk of respiratory infections.

An overview of the role of platelets in angiogenesis, apoptosis and autophagy in chronic myeloid leukaemia.

Amongst males, leukaemia is the most common cause of cancer-related death in individuals younger than 40 years of age whereas in female children and adolescents, leukaemia is the most common cause of cancer-related death. Chronic myeloid leukaemia (CML) is a chronic leukaemia of the haematopoietic stem cells affecting mostly adults. The disease results from a translocation of the Philadelphia chromosome in stem cells of the bone marrow. CML patients usually present with mild to moderate anaemia and with decreased, normal, or increased platelet counts. CML represents 0.5% of all new cancer cases in the United States (2016). In 2016, an estimated 1070 people would die of this disease in the United States. Platelets serve as a means for tumours to increase growth and to provide physical- and mechanical support to elude the immune system and to metastasize. Currently there is no literature available on the role that platelets play in CML progression, despite literature reporting the fact that platelet count and size are affected. Resistance to CML treatment with tyrosine kinase inhibitors can be as a result of acquired resistance ensuing from mutations in the tyrosine kinase domains, loss of response or poor tolerance. In CML this resistance has recently become linked to bone marrow (BM) angiogenesis which aids in the growth and survival of leukaemia cells. The discovery of the lungs as a site of haematopoietic progenitors, suggests that CML resistance is not localized to the bone marrow and that the mutations leading to the disease and resistance to treatment may also occur in the haematopoietic progenitors in the lungs. In conclusion, platelets are significantly affected during CML progression and treatment. Investigation into the role that platelets play in CML progression is vital including how treatment affects the cell death mechanisms of platelets.

Pneumolysin mediates heterotypic aggregation of neutrophils and platelets in vitro.

OBJECTIVES: Platelets orchestrate the inflammatory activities of neutrophils, possibly contributing to pulmonary and myocardial damage during severe pneumococcal infection. This study tested the hypothesis that the pneumococcal toxin, pneumolysin (Ply), activates production of platelet-activating factor (PAF) and thromboxane A2 (TxA2) by neutrophils, these bioactive lipids being potential mediators of neutrophil:platelet (NP) networking. METHODS: The effects of recombinant Ply (10-80 ng mL-1) on the production of PAF and TxA2 by isolated neutrophils were measured using ELISA procedures, and NP aggregation by flow cytometry. RESULTS: Exposure of neutrophils to Ply induced production of PAF and, to a lesser extent, TxA2, achieving statistical significance at ≥20 ng mL-1 of the toxin. In the case of NP interactions, Ply promoted heterotypic aggregation which was dependent on upregulation of P-selectin (CD62P) and activation of protease-activated receptor 1 (PAR1), attaining statistical significance at ≥10 ng mL-1 of the toxin, but did not involve either PAF or TxA2. CONCLUSION: Ply induces synthesis of PAF and TxA2, by human neutrophils, neither of which appears to contribute to the formation of NP heterotypic aggregates in vitro, a process which is seemingly dependent on CD62P and PAR1. These pro-inflammatory activities of Ply may contribute to the pathogenesis of pulmonary and myocardial injury during severe pneumococcal infection.

Detection of Acute and Early HIV-1 Infections in an HIV Hyper-Endemic Area with Limited Resources.

BACKGROUND: Two thirds of the world's new HIV infections are in sub-Saharan Africa. Acute HIV infection (AHI) is the time of virus acquisition until the appearance of HIV antibodies. Early HIV infection, which includes AHI, is the interval between virus acquisition and establishment of viral load set-point. This study aimed to detect acute and early HIV infections in a hyper-endemic setting. METHODS: This was a cross-sectional diagnostic study that enrolled individuals who had negative rapid HIV results in five clinics in South Africa. Pooled nucleic acid amplification testing (NAAT) was performed, followed by individual sample testing in positive pools. NAAT-positive participants were recalled to the clinics for confirmatory testing and appropriate management. HIV antibody, p24 antigen, Western Blot and avidity tests were performed for characterization of NAAT-positive samples. RESULTS: The study enrolled 6910 individuals with negative rapid HIV results. Median age was 27 years (interquartile range {IQR}: 23-31). NAAT was positive in 55 samples, resulting in 0.8% newly diagnosed HIV-infected individuals (95% confidence interval {CI}: 0.6-1.0). The negative predictive value for rapid HIV testing was 99.2% (95% CI: 99.0-99.4). Characterization of NAAT-positive samples revealed that 0.04% (95% CI: 0.000-0.001) had AHI, 0.3% (95% CI: 0.1-0.4) had early HIV infection, and 0.5% (95% CI: 0.5-0.7) had chronic HIV infection. Forty-seven (86%) of NAAT-positive participants returned for follow-up at a median of 4 weeks (IQR: 2-8). Follow-up rapid tests were positive in 96% of these participants. CONCLUSIONS: NAAT demonstrated that a substantial number of HIV-infected individuals are misdiagnosed at South African points-of-care. Follow-up rapid tests done within a 4 week interval detected early and chronic HIV infections initially missed by rapid HIV testing. This may be a practical and affordable strategy for earlier detection of these infections in resource-constrained settings. Newer molecular tests that can be used at the points-of-care should be evaluated for routine diagnosis of HIV in hyper-endemic settings.

Pneumolysin Mediates Platelet Activation In Vitro.

This study has explored the role of the pneumococcal toxin, pneumolysin (Ply), in activating human platelets. Following exposure to Ply (10-80 ng/ml), platelet activation and cytosolic Ca(2+) concentrations were measured flow cytometrically according to the level of expression of CD62P (P-selectin) and spectrofluorimetrically, respectively. Exposure to Ply resulted in marked upregulation of expression of platelet CD62P, achieving statistical significance at concentrations of 40 ng/ml and higher (P < 0.05), in the setting of increased influx of Ca(2+). These potentially pro-thrombotic actions of Ply were attenuated by depletion of Ca(2+) from the extracellular medium or by exposure of the cells to a pneumolysoid devoid of pore-forming activity. These findings are consistent with a mechanism of Ply-mediated platelet activation involving sub-lytic pore formation, Ca(2+) influx, and mobilization of CD62P-expressing α-granules, which, if operative in vivo, may contribute to the pathogenesis of associated acute lung and myocardial injury during invasive pneumococcal disease.