Replacement of Antiretroviral Therapy with HIV Broadly Neutralizing Antibodies to Maximize the Effectiveness of Chemotherapy in HIV Patients with Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most fatal non-AIDS defining cancer in people living with HIV (PWH) on antiretroviral therapy (ART). Treatment of malignancies in PWH requires concomitant cancer therapy and ART, which can lead to potential drug-drug interactions (DDIs) and overlapping toxicities. In this study, we hypothesize that replacement of ART with HIV broadly neutralizing antibodies (bNAbs) during cancer chemotherapy (chemo) may maintain HIV suppression and tumor inhibition while minimizing DDIs and overlapping toxicities. We compared HIV suppression, tumor inhibition, and toxicity between conventional treatment (ART plus chemo) and a new modality (bNAbs plus chemo) in humanized mice. Humanized mice infected with HIVYU2 and xenografted with human NSCLC A549 cells were treated with NSCLC chemo (cisplatin and gemcitabine) and first-line ART (dolutegravir, tenofovir disoproxil difumarate, and emtricitabine) or bNAbs (N49P9.6-FR and PGT 121) at human equivalent drug doses. We monitored plasma HIV RNA, tumor volume, and toxicities over five cycles of chemo. We found that chemo plus ART or bNAbs were equally effective at maintaining suppression of HIV viremia and tumor growth. Comparative analysis showed that mice on ART and chemo had significant reductions in body weight and significant increases in plasma creatinine concentrations compared with mice on bNAbs and chemo, which suggests that a combination of bNAbs and chemo produces less renal toxicity than an ART and chemo combination. These data suggest that bNAb therapy during concomitant chemo may be an improved treatment option over ART for PWH and NSCLC, and possibly other cancers, because bNAbs maintain HIV suppression while minimizing DDIs and toxicities.

Exploring the Role of Virtual Reality to Support Clinical Diabetes Training-A Pilot Study.

BACKGROUND: It is estimated that 16 to 25% of patients in hospital have diabetes and 1 in 25 inpatients with Type 1 Diabetes develop diabetic ketoacidosis (DKA). It is vital that non-specialist doctors recognize and appropriately manage diabetes emergencies. Simulation training is increasingly being used in healthcare and virtual reality (VR) based educational resources is transforming medical education. This study aimed to evaluate the use of virtual reality to help non-specialist clinicians manage clinical scenarios related to diabetes. METHODS: This pilot project, titled 'DEVICE' (Diabetes Emergencies: Virtual Interactive Clinical Education) was developed in collaboration with Oxford Medical Simulation. Fully interactive immersive VR scenarios were created to stimulate real life diabetes emergencies. Users then received personalized feedback and performance metrics. Feedback surveys were provided before and after the participation in the VR scenario. Kirkpatrick's training evaluation model was used. RESULTS: Thirty-nine participants from 2 hospitals in UK provided feedback up to 3 months after attending the VR education sessions. Overall feedback was extremely positive, and participants found this immersive teaching experience very helpful. After use of virtual reality scenarios, the mean trainee confidence in managing DKA (on an 8-point Likert scale) increased from 3.92 (3.38-4.47) 95% CI to 5.41 (4.79-6.03) 95% CI (statistically significant). The VR study demonstrates Kirkpatrick level 3 in the follow up survey. CONCLUSION: VR based training scenarios in this pilot project increased confidence in managing diabetes emergencies and demonstrated positive changes in their behavior. VR education is a safe, useful and a well-liked training tool for diabetes emergencies.

Evaluating the Impact of Inadequate Meal Consumption on Insulin-Related Hypoglycemia in Hospitalized Patients.

OBJECTIVE: Meal intake is sometimes reduced in hospitalized patients. Meal-time insulin administration can cause hypoglycemia when a meal is not consumed. Inpatient providers may avoid ordering meal-time insulin due to hypoglycemia concerns, which can result in hyperglycemia. The frequency of reduced meal intake in hospitalized patients remains inadequately determined. This quality improvement project evaluates the percentage of meals consumed by hospitalized patients with insulin orders and the resulting risk of postmeal hypoglycemia (blood glucose [BG] <70 mg/dL, <3.9 mmol/L). METHODS: This was a retrospective quality improvement project evaluating patients with any subcutaneous insulin orders hospitalized at a regional academic medical center between 2015 and 2017. BG, laboratory values, point of care, insulin administration, diet orders, and percentage of meal consumed documented by registered nurses were abstracted from electronic health records. RESULTS: Meal consumption ≥50% was observed for 85% of meals with insulin orders, and bedside registered nurses were accurate at estimating this percentage. Age ≥65 years was a risk factor for reduced meal consumption (21% of meals 0%-49% consumed, P < .05 vs age < 65 years [12%]). Receiving meal-time insulin and then consuming only 0% to 49% of a meal (defined here as a mismatch) was not rare (6% of meals) and increased postmeal hypoglycemia risk. However, the attributable risk of postmeal hypoglycemia due to this mismatch was low (4 events per 1000) in patients with premeal BG between 70 and 180 mg/dL. CONCLUSION: This project demonstrates that hospitalized patients treated with subcutaneous insulin have a low attributable risk of postmeal hypoglycemia related to inadequate meal intake.

Apple polyphenol-rich drinks dose-dependently decrease early-phase postprandial glucose concentrations following a high-carbohydrate meal: a randomized controlled trial in healthy adults and in vitro studies.

BACKGROUND: Previous research demonstrated that a high dose of phlorizin-rich apple extract (AE) can markedly inhibit early-phase postprandial glycemia, but efficacy of lower doses of the AE is unclear. OBJECTIVE: To determine whether lower AE doses reduce early-phase postprandial glycemia in healthy adults and investigate mechanisms. DESIGN: In a randomized, controlled, double-blinded, cross-over acute trial, drinks containing 1.8 g (HIGH), 1.35 g (MED), 0.9 g (LOW), or 0 g (CON) of a phlorizin-rich AE were consumed before 75 g starch/sucrose meal. Postprandial blood glucose, insulin, C-peptide, glucose-dependent insulinotropic polypeptide (GIP) and polyphenol metabolites concentrations were measured 0-240 min, acetaminophen concentrations to assess gastric emptying rate, and 24 h urinary glucose excretion. Effects of AE on intestinal glucose transport were investigated in Caco-2/TC7 cells. RESULTS: AE significantly reduced plasma glucose iAUC 0-30 min at all doses: mean differences (95% CI) relative to CON were -15.6 (-23.3, -7.9), -11.3 (-19.6, -3.0) and -8.99 (-17.3, -0.7) mmol/L per minute for HIGH, MEDIUM and LOW respectively, delayed Tmax (HIGH, MEDIUM and LOW 45 min vs. CON 30 min), but did not lower Cmax. Similar dose-dependent treatment effects were observed for insulin, C-peptide, and GIP. Gastric emptying rates and urinary glucose excretion did not differ. Serum phloretin, quercetin and epicatechin metabolites were detected postprandially. A HIGH physiological AE dose equivalent decreased total glucose uptake by 48% in Caco-2/TC7 cells. CONCLUSIONS: Phlorizin-rich AE, even at a low dose, can slightly delay early-phase glycemia without affecting peak and total glycemic response.

Fructosamine and Hemoglobin A1c Correlations in HIV-Infected Adults in Routine Clinical Care: Impact of Anemia and Albumin Levels.

Fructosamine is an alternative method to hemoglobin A1c (HbA1c) for determining average glycemia. However, its use has not been extensively evaluated in persons living with HIV (PLWH). We examined the relationship between HbA1c and fructosamine values, specifically focusing on anemia (which can affect HbA1c) and albumin as a marker of liver disease. We included 345 PLWH from two sites. We examined Spearman rank correlations between fructosamine and HbA1c and performed linear test for trends to compare fructosamine and HbA1c correlations by hemoglobin and albumin quartiles. We examined discrepant individuals with values elevated only on one test. We found a correlation of 0.70 between fructosamine and HbA1c levels. Trend tests for correlations between fructosamine and HbA1c were significant for both albumin (p = 0.05) and hemoglobin (p = 0.01) with the lowest correlations in the lowest hemoglobin quartile. We identified participants with unremarkable HbA1c values but elevated fructosamine values. These discrepant individuals had lower mean hemoglobin levels than those elevated by both tests. We demonstrated a large correlation between HbA1c and fructosamine across a range of hemoglobin and albumin levels. There were discrepant cases particularly among those with lower hemoglobin levels. Future studies are needed to clarify the use of fructosamine for diabetes management in PWLH.

Laser-induced endothelial cell activation supports fibrin formation.

Laser-induced vessel wall injury leads to rapid thrombus formation in an animal thrombosis model. The target of laser injury is the endothelium. We monitored calcium mobilization to assess activation of the laser-targeted cells. Infusion of Fluo-4 AM, a calcium-sensitive fluorochrome, into the mouse circulation resulted in dye uptake in the endothelium and circulating hematopoietic cells. Laser injury in mice treated with eptifibatide to inhibit platelet accumulation resulted in rapid calcium mobilization within the endothelium. Calcium mobilization correlated with the secretion of lysosomal-associated membrane protein 1, a marker of endothelium activation. In the absence of eptifibatide, endothelium activation preceded platelet accumulation. Laser activation of human umbilical vein endothelial cells loaded with Fluo-4 resulted in a rapid increase in calcium mobilization associated cell fluorescence similar to that induced by adenosine diphosphate (10 µM) or thrombin (1 U/mL). Laser activation of human umbilical vein endothelial cells in the presence of corn trypsin inhibitor treated human plasma devoid of platelets and cell microparticles led to fibrin formation that was inhibited by an inhibitory monoclonal anti-tissue factor antibody. Thus laser injury leads to rapid endothelial cell activation. The laser activated endothelial cells can support formation of tenase and prothrombinase and may be a source of activated tissue factor as well.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation.

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Critical role of FcR gamma-chain, LAT, PLCgamma2 and thrombin in arteriolar thrombus formation upon mild, laser-induced endothelial injury in vivo.

OBJECTIVE: The role of collagen receptor complex GPVI-FcR gamma-chain, PLCgamma2 and LAT in laser-induced thrombosis is unclear. Controversy surrounds whether collagen is exposed in this model or whether thrombosis is dependent on thrombin. This study hypothesized that collagen exposure plays a critical role in thrombus formation in this model, which was tested by investigating contributions of FcR gamma-chain, LAT, PLCgamma2 and thrombin. METHODS: Thrombi were monitored using intravital microscopy in anesthetized wild-type and FcR gamma-chain, LAT and PLCgamma2 knockout mice. Hirudin (thrombin inhibitor) was administered to wild-type and FcR gamma-chain knockout mice. RESULTS: Significantly reduced thrombus formation was observed in FcR gamma-chain and PLCgamma2 knockouts with a greater decrease observed in LAT knockouts. Dramatic reduction was observed in wild-types treated with hirudin, with abolished thrombus formation only observed in FcR gamma-chain knockouts treated with hirudin. CONCLUSIONS: GPVI-FcR gamma-chain, LAT and PLCgamma2 are essential for thrombus generation and stability in this laser-induced model of injury. More importantly, a greater role for LAT was identified, which may reflect a role for it downstream of a second matrix protein receptor. However, inhibition of platelet activation by matrix proteins and thrombin generation are both required to maximally prevent thrombus formation.

Ecto-nucleotidases of the CD39/NTPDase family modulate platelet activation and thrombus formation: Potential as therapeutic targets.

Extracellular nucleotide P2-receptor-mediated effects on platelets, leukocytes and endothelium are modulated by ecto-nucleotidases. These ecto-enzymes hydrolyze extracellular nucleotides to the respective nucleosides. The dominant ecto-nucleotidase expressed by the endothelium, by monocytes and vascular smooth muscle cells is CD39/NTPDase1. Ecto-nucleotidase biochemical activity of CD39 is lost at sites of acute vascular injury, such as in ischemia reperfusion and immune graft rejection. CD39L(Like)1/NTPDase2, a related protein, is associated with the basolateral surface of endothelium, the adventitia of vessels and microvascular pericytes. CD39/NTPDase1 hydrolyzes both tri- and diphosphonucleosides and blocks platelet aggregation responses to ADP. In contrast, CD39L1/NTPDase2, a preferential nucleoside triphosphatase, activates platelets by preferentially converting ATP to ADP, the major agonist of platelet P2 receptors. Spatial and temporal expression of NTPDases in the vasculature appears to control platelet activation, thrombus size and stability by regulating phosphohydrolytic activity and consequent P2 receptor signaling. Constitutively circulating microparticles appear to be associated with functional NTPDases, and accumulation of these at sites of vascular injury might influence local thrombus formation and evolution. The phenotype of the cd39-null mouse is in keeping with disordered thromboregulation with heightened susceptibility to inflammatory vasculary reactions, increased permeability and high levels of tissue fibrin. Paradoxically, these mutant mice also exhibit a bleeding phenotype with differential platelet P2Y1 desensitization. Over-expression of CD39 at sites of vascular injury and inflammation by adenoviral vectors, by transgenesis or by the use of pharmacological modalities with soluble derivatives has been shown to have major potential in several animal models tested to date. Future clinical applications will involve the development of new therapeutic strategies to various inflammatory vascular diseases and in transplantation.

Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow.

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo.

Role of the p110delta PI 3-kinase in integrin and ITAM receptor signalling in platelets.

We have investigated the function of the p110delta catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in platelets using p110delta knock-out (p110delta(-/-)) mice and p110delta knock-in (p110delta(D910A/D910A)) mice, which express a catalytically inactive form of the enzyme. Aggregation to threshold concentrations of the GPVI-specific agonist, CRP, was partially reduced in p110delta(-/-) and p110delta(D910A/D910A) platelets. This inhibition was overcome by higher concentrations of CRP. The degree of inhibition was considerably weaker than that induced by LY294002 and wortmannin, which inhibit all isoforms of PI 3-kinase. p110delta(-/-) platelets showed decreased spreading on fibrinogen- or von Willebrand factor (VWF)-coated surfaces under static conditions, whereas they spread normally on collagen. LY294002 had a more pronounced inhibitory effect on spreading on all three surfaces. Adhesion and aggregate formation of p110delta(-/-) platelets to collagen or fibrinogen/VWF at intermediate/high rates of shear were normal. This study demonstrates a minor role for the p110delta catalytic subunit in mediating platelet activation by the collagen receptor GPVI and integrin alphaIIbeta3. The more pronounced inhibitory effect of LY294002 and wortmannin indicates that other isoforms of PI 3-kinase play a more significant role in signalling by the two platelet glycoprotein receptors.

Tec regulates platelet activation by GPVI in the absence of Btk.

The Tec family kinase Btk plays an important role in the regulation of phospholipase C gamma 2 (PLC gamma 2) downstream of the collagen receptor glycoprotein VI (GPVI) in human platelets. Platelets also express a second member of this family, Tec; however, its function has not been analyzed. To address the role of Tec, we analyzed Btk-/-, Tec-/-, and Btk/Tec double-deficient (Btk-/-/Tec-/-) platelets. Tec-/- platelets exhibit a minor reduction in aggregation to threshold concentrations of collagen or the GPVI-specific agonist collagen-related peptide (CRP), whereas responses to higher concentrations are normal. Tyrosine phosphorylation of PLC gamma 2 by collagen and CRP is not altered in Tec-/- platelets. However, Btk-/-/Tec-/- platelets exhibit a greater reduction in PLC gamma 2 phosphorylation than is seen in the absence of Btk, thus revealing an important role for Tec in this situation. Furthermore, Btk-/-/Tec-/- platelets fail to undergo an increase in Ca2+, aggregation, secretion, and spreading in response to collagen or CRP, whereas they aggregate normally to adenosine diphosphate (ADP) and spread on fibrinogen. A residual GPVI signal exists in the Btk-/-/Tec-/- platelets as CRP synergizes with ADP to mediate aggregation. These results demonstrate an essential requirement for Tec and Btk in platelet activation by GPVI and reveal a functional role for Tec in the regulation of PLC gamma 2 in the absence of Btk.

Thrombin-induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb.

1. Activation of human platelets by thrombin is mediated by the proteolytic cleavage of two G-protein coupled protease-activated receptors, PAR-1 and PAR-4. However, thrombin also binds specifically to the platelet surface glycoprotein GPIb. It has been claimed that thrombin can induce aggregation of platelets via a novel GPIb-mediated pathway, which is independent of PAR activation and fibrinogen binding to alpha(IIb)beta(3) integrin, but dependent upon polymerizing fibrin and the generation of intracellular signals. 2. In the presence of both fibrinogen and the alpha(IIb)beta(3) receptor antagonist lotrafiban, thrombin induced a biphasic platelet aggregation response. The initial primary response was small but consistent and associated with the release of platelet granules. The delayed secondary response was more substantial and was abolished by the fibrin polymerization blocking peptide GPRP. 3. Cleavage of the extracellular portion of GPIb by mocarhagin partially inhibited thrombin-induced alpha(IIb)beta(3)-dependent aggregation and release, but had no effect on the secondary fibrin-dependent response. 4. Fixing of the platelets abolished alpha(IIb)beta(3)-dependent aggregation and release of adenine nucleotides, whereas the fibrin-dependent response remained, indicating that platelet activation and intracellular signalling are not necessary for this secondary 'aggregation'. 5. In conclusion, the secondary fibrin-dependent 'aggregation' response observed in the presence of fibrinogen and lotrafiban is a platelet trapping phenomenon dependent primarily on the conversion of soluble fibrinogen to polymerizing fibrin by thrombin.

Distinct roles of GPVI and integrin alpha(2)beta(1) in platelet shape change and aggregation induced by different collagens.

1. Various platelet membrane glycoproteins have been proposed as receptors for collagen, in some cases as receptors for specific collagen types. In this study we have compared the ability of a range of collagen types to activate platelets. 2. Bovine collagen types I-V, native equine tendon collagen fibrils and collagen-related peptide (CRP) all induced platelet aggregation and shape change. 3. Responses were abolished in FcRgamma chain-deficient platelets, which also lack GPVI, indicating a critical dependence on the GPVI/FcRgamma chain complex. 4. Responses to all collagens were unaffected in CD36-deficient platelets. 5. A monoclonal antibody (6F1) which binds to the alpha(2) integrin subunit of human platelets had a minimal effect on the rate and extent of aggregation induced by the collagens; however, it delayed the onset of aggregation following addition of all collagens. For shape change, 6F1 abolished the response induced by collagen types I and IV, substantially attenuated that to collagen types II, III and V, but only partially inhibited Horm collagen. 6. Simultaneous blockade of the P2Y(1) and P2Y(12) receptors, and inhibition of cyclo-oxygenase demonstrated that CRP can activate platelets independently of ADP and TxA(2); however, responses to the collagens were dependent on these mediators. 7. This study confirms the importance of the GPVI/FcRgamma chain complex in platelet responses induced by a range of collagen agonists, while providing no evidence for collagen type-specific receptors. It also provides evidence for a modulatory role of alpha(2)beta(1), the significance of which depends on the collagen preparation.