Quantum Dot Imaging Agents: Haematopoietic Cell Interactions and Biocompatibility.

Quantum dots (QDs) are semi-conducting nanoparticles that have been developed for a range of biological and non-biological functions. They can be tuned to multiple different emission wavelengths and can have significant benefits over other fluorescent systems. Many studies have utilised QDs with a cadmium-based core; however, these QDs have since been shown to have poor biological compatibility. Therefore, other QDs, such as indium phosphide QDs, have been developed. These QDs retain excellent fluorescent intensity and tunability but are thought to have elevated biological compatibility. Herein we discuss the applicability of a range of QDs to the cardiovascular system. Key disease states such as myocardial infarction and stroke are associated with cardiovascular disease (CVD), and there is an opportunity to improve clinical imaging to aide clinical outcomes for these disease states. QDs offer potential clinical benefits given their ability to perform multiple functions, such as carry an imaging agent, a therapy, and a targeting motif. Two key cell types associated with CVD are platelets and immune cells. Both cell types play key roles in establishing an inflammatory environment within CVD, and as such aid the formation of pathological thrombi. However, it is unclear at present how and with which cell types QDs interact, and if they potentially drive unwanted changes or activation of these cell types. Therefore, although QDs show great promise for boosting imaging capability, further work needs to be completed to fully understand their biological compatibility.

Impact of Surface Ligand on the Biocompatibility of InP/ZnS Quantum Dots with Platelets.

InP/ZnS quantum dots (QDs) have received a large focus in recent years as a safer alternative to heavy metal-based QDs. Given their intrinsic fluorescent imaging capabilities, these QDs can be potentially relevant for in vivo platelet imaging. The InP/ZnS QDs are synthesized and their biocompatibility investigated through the use of different phase transfer agents. Analysis of platelet function indicates that platelet-QD interaction can occur at all concentrations and for all QD permutations tested. However, as the QD concentration increases, platelet aggregation is induced by QDs alone independent of natural platelet agonists. This study helps to define a range of concentrations and coatings (thioglycolic acid and penicillamine) that are biocompatible with platelet function. With this information, the platelet-QD interaction can be identified using multiple methods. Fluorescent lifetime imaging microscopy (FLIM) and confocal studies have shown QDs localize on the surface of the platelet toward the center while showing evidence of energy transfer within the QD population. It is believed that these findings are an important stepping point for the development of fluorescent probes for platelet imaging.

Platelet zinc status regulates prostaglandin-induced signaling, altering thrombus formation.

BACKGROUND: Approximately 17.3% of the global population exhibits an element of zinc (Zn2+) deficiency. One symptom of Zn2+ deficiency is increased bleeding through impaired hemostasis. Platelets are crucial to hemostasis and are inhibited by endothelial-derived prostacyclin (prostaglandin I2 [PGI2]), which signals via adenylyl cyclase (AC) and cyclic adenosine monophosphate signaling. In other cell types, Zn2+ modulates cyclic adenosine monophosphate concentrations by changing AC and/or phosphodiesterase activity. OBJECTIVES: To investigate if Zn2+ can modulate platelet PGI2 signaling. METHODS: Platelet aggregation, spreading, and western blotting assays with Zn2+ chelators and cyclic nucleotide elevating agents were performed in washed platelets and platelet-rich plasma conditions. In vitro thrombus formation with various Zn2+ chelators and PGI2 was assessed in whole blood. RESULTS: Incubation of whole blood or washed platelets with Zn2+ chelators caused either embolization of preformed thrombi or reversal of platelet spreading, respectively. To understand this effect, we analyzed resting platelets and identified that incubation with Zn2+ chelators elevated pVASPser157, a marker of PGI2 signaling. In agreement that Zn2+ affects PGI2 signaling, addition of the AC inhibitor SQ22536 blocked Zn2+ chelation-induced platelet spreading reversal, while addition of Zn2+ blocked PGI2-mediated platelet reversal. Moreover, Zn2+ specifically blocked forskolin-mediated AC reversal of platelet spreading. Finally, PGI2 inhibition of platelet aggregation and in vitro thrombus formation was potentiated in the presence of low doses of Zn2+ chelators, increasing its effectiveness in inducing platelet inhibition. CONCLUSION: Zn2+ chelation potentiates platelet PGI2 signaling, elevating PGI2's ability to prevent effective platelet activation, aggregation, and thrombus formation.

Bruton's Tyrosine Kinase Inhibitors Impair FcγRIIA-Driven Platelet Responses to Bacteria in Chronic Lymphocytic Leukemia.

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton's tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbß3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro. While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbß3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions.

Practical Considerations of Dissolved Oxygen Levels for Platelet Function under Hypoxia.

Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 108/mL versus 2 × 107/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.

Central activation deficits contribute to post stroke lingual weakness in a rat model.

Lingual weakness frequently occurs after stroke and is associated with deficits in speaking and swallowing. Chronic weakness after stroke has been attributed to both impaired central activation of target muscles and reduced force-generating capacity within muscles. How these factors contribute to lingual weakness is not known. We hypothesized that lingual weakness due to middle cerebral artery occlusion (MCAO) would manifest as reduced muscle force capacity and reduced muscle activation. Rats were randomized into MCAO or sham surgery groups. Maximum volitional tongue forces were quantified 8 wk after surgery. Hypoglossal nerve stimulation was used to assess maximum stimulated force, muscle twitch properties, and force-frequency response. The central activation ratio was determined by maximum volitional/maximum stimulated force. Genioglossus muscle fiber type properties and neuromuscular junction innervation were assessed. Maximum volitional force and the central activation ratio were significantly reduced with MCAO. Maximum stimulated force was not significantly different. No significant differences were found for muscle twitch properties, unilateral contractile properties, muscle fiber type percentages, or fiber size. However, the twitch/tetanus ratio was significantly increased in the MCAO group relative to sham. A small but significant increase in denervated neuromuscular junctions (NMJs) and fiber-type grouping occurred in the contralesional genioglossus. Results suggest that the primary cause of chronic lingual weakness after stroke is impaired muscle activation rather than a deficit of force-generating capacity in lingual muscles. Increased fiber type grouping and denervated NMJs in the contralesional genioglossus suggest that partial reinnervation of muscle fibers may have preserved force-generating capacity, but not optimal activation patterns.NEW & NOTEWORTHY Despite significant reductions in maximum volitional forces, the intrinsic force-generating capacity of the protrusive lingual muscles was not reduced with unilateral cerebral ischemia. Small yet significant increases in denervated NMJs and fiber-type grouping of the contralesional genioglossus suggest that the muscle underwent denervation and reinnervation. Together these results suggest that spontaneous neuromuscular plasticity was sufficient to prevent atrophy, yet central activation deficits remain and contribute to chronic lingual weakness after stroke.

A service evaluation of an integrated model of palliative care of cystic fibrosis.

BACKGROUND: Patients with advanced cystic fibrosis have severe symptoms with a complex trajectory of exacerbations and recovery. They are often awaiting lung transplantation, and many die without receiving specialist palliative care. AIM: We introduced an integrated model whereby palliative specialists joined the cystic fibrosis team to provide palliative care in parallel with standard care. DESIGN: A service evaluation of this model of care was undertaken in a prospective case series documenting symptoms and outcomes, the views of the cystic fibrosis team and the experience of the palliative specialists. SETTING/PARTICIPANTS: Over 3 years, 28 (10%) of 282 patients attending the cystic fibrosis centre had specialist palliative care. RESULTS: They had advanced lung disease (mean forced expiratory volume in 1 s (FEV1) = 0.86 L (25% predicted)), and 17 died: 6 were on a transplant waiting list at death; 10 were unsuitable and 1 died post transplantation. All who died over these 3 years had specialist palliative care. Four patients had successful transplants. Assessment showed a high prevalence of breathlessness, cough, pain, vomiting and fatigue, with a significant impact on daily life. The cystic fibrosis team rated this model of care highly, felt that palliative care should be members of the team, and thought that patients had found it helpful. The palliative specialists gained knowledge of cystic fibrosis, found it beneficial to meet patients earlier in the disease, and identified unmet needs in managing bereavement and the effects of deaths on other patients with cystic fibrosis. CONCLUSION: This model has been successful in overcoming the difficulties in access to specialist palliative care for patients with cystic fibrosis.