Organotypic endothelial adhesion molecules are key for Trypanosoma brucei tropism and virulence.

Trypanosoma brucei is responsible for lethal diseases in humans and cattle in Sub-Saharan Africa. These extracellular parasites extravasate from the blood circulation into several tissues. The importance of the vasculature in tissue tropism is poorly understood. Using intravital imaging and bioluminescence, we observe that gonadal white adipose tissue and pancreas are the two main parasite reservoirs. We show that reservoir establishment happens before vascular permeability is compromised, suggesting that extravasation is an active mechanism. Blocking endothelial surface adhesion molecules (E-selectin, P-selectins, or ICAM2) significantly reduces extravascular parasite density in all organs and delays host lethality. Remarkably, blocking CD36 has a specific effect on adipose tissue tropism that is sufficient to delay lethality, suggesting that establishment of the adipose tissue reservoir is necessary for parasite virulence. This work demonstrates the importance of the vasculature in a T. brucei infection and identifies organ-specific adhesion molecules as key players for tissue tropism.

Selectin Dependence of Allergic Skin Inflammation Is Diminished by Maternal Atopy.

Allergic skin inflammation requires the influx of inflammatory cells into the skin. Extravasation of leukocytes into the skin requires interactions between endothelial selectins and their glycan ligands on the surface of leukocytes. Selectin-ligand formation requires the activity of several glycosyltransferases, including Fut7 In this report, we tested the importance of Fut7 for the development of allergic skin inflammation in the Stat6VT transgenic mouse model. We observed that Fut7 deficiency was protective but did not eliminate disease. Segregation of the data by gender of the parent that transmitted the Stat6VT transgene, but not by gender of the pups, which were analyzed for disease, revealed that the protective effects of Fut7 deficiency were significantly greater when dams were Stat6VT negative. In contrast, in mice from litters of Stat6VT+ dams, Fut7 deficiency resulted in only modest protection. These findings indicate that pups from atopic dams exhibit a greater propensity for allergic disease, similar to observations in humans, and that the effect of maternal atopy is due to enhanced selectin-independent mechanisms of leukocyte recruitment in their offspring. Together, these results demonstrate that Fut7 deficiency can be protective in a model of atopic dermatitis but that maternal atopy diminishes these protective effects, suggesting alternative pathways for leukocyte recruitment in the absence of Fut7 enzyme activity. These observations have implications for understanding how the environment in utero predisposes for the development of allergic disease.

A prospective, blinded study of a PF4-dependent assay for HIT diagnosis.

Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14-labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.

Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes.

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.

CD226 is involved in megakaryocyte activation and early-stage differentiation.

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.

The changes in immune cell concentration during the progression of pre-diabetes to type 2 diabetes in a high-fat high-carbohydrate diet-induced pre-diabetic rat model.

Pre-diabetes is a long-lasting condition that precedes type 2 diabetes (T2D). T2D has been shown to suppress the immune response. However, it remains unclear if immune activation occurs before the onset of T2D during the progression of the pre-diabetic state. This study sought to characterize the changes in general immunity occurring during the progression from pre-diabetes to T2D. Male rats were fed a high-fat high-carbohydrate diet for 20 weeks (pre-diabetes induction period) and kept on the same diet being monitored for a further 12 weeks (experimental period). Blood was collected for haemocytometer analysis on week 0, 4, 8, and 12 of the experimental period after which the animals were sacrificed. Plasma was collected from centrifuged blood for ELISA (TNF-α, CRP, P-selectin, CD40 L, fibrinogen, and IL-6). Blood neutrophils percentage significantly decreased at week 12 possibly due to recruited neutrophils migrating to an inflamed area such as visceral adipose tissue as further observed. Due to hyperglycaemia, there was significant increase in blood lymphocytes percentage at week 12. Blood monocytes percentage significantly increased at week 12. Monocytes recruited and circulated in blood due to hyperglycaemia for glucose uptake to decrease it from circulation. Blood eosinophils percentage significantly decreased at week 12. Eosinophils migrated to inflamed areas such as visceral adipose tissue as further observed. Blood basophils percentage significantly increased due to their recruitment and activation. TNF-α, CRP, and IL-6 increased significantly after 12 weeks. There was also upregulation of fibrinogen, P-selectin, and CD40L. The results of this study show that there are changes in immune cells concentration and that immune cells such as neutrophils and eosinophils migrate to inflamed areas such as adipose tissue. There is also upregulation of various inflammatory cytokines. Based on these findings, immune activation begins during the pre-diabetic state as there is upregulation of inflammatory markers.

Emerging pharmacotherapeutic approaches for the management of sickle cell disease.

INTRODUCTION: Sickle cell disease (SCD) is an inherited disease with lifelong morbidity, whose complications include frequent acute painful vaso-occlusive episodes (VOEs) that often require hospitalization. The only pharmacotherapy currently in regular use for SCD management is hydroxyurea (hydroxycarbamide). AREAS COVERED: We review recent advances in pharmacotherapy for SCD and summarize promising synthetic agents that are in late-stage development (phase 3) for SCD. EXPERT OPINION: Emerging SCD therapies have been developed to target specific pathophysiological mechanisms of the disease, as either preventative or abortive approaches to VOEs. Continuous-use pharmacotherapeutics in late-phase development for VOE prevention include voxelotor (GBT440), which elevates hemoglobin oxygenation, and prasugrel, a platelet activation inhibitor. However, at least in the near future, it is probable that biological molecules will play a primary role in SCD preventative therapy; in combination with hydroxyurea, crizanlizumab, an anti-P-selectin monoclonal antibody, appears to reduce VOE frequency, while L-glutamine was the first substance licensed by the FDA for use in SCD in 20 years. Synthetic drugs, however, may represent key approaches for the management of individuals upon hospitalization for VOE, a major challenge for SCD. For example, rivipansel (GMI-1070), a pan-selectin inhibitor, has shown encouraging effects on hospitalization time and opioid use.

Critical role of C5a in sickle cell disease.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.

P-Selectin Is Critical for De Novo Pulmonary Arterial Thrombosis Following Blunt Thoracic Trauma.

BACKGROUND: Thromboembolic events within the pulmonary arterial vasculature are a troublesome complication of severe blunt thoracic trauma. Mechanisms underlying these events are currently in question as pulmonary thromboembolic events in this particular trauma population tend to be diagnosed more rapidly, more frequently and without an associated systemic thrombosis. This study investigates the role of P-selectin in thrombus formation through the use of in vivo blocking antibodies. We hypothesize that P-selectin plays a pivotal role in de novo pulmonary arterial thrombosis following blunt thoracic trauma. METHODS: A murine weight-drop model of lateral blunt thoracic trauma was used. Wild-type mice in the experimental group were given blocking antibodies against P-selectin prior to the trauma. All mice were euthanized at 24 hours for evaluation with hematoxylin-eosin staining or immunofluorescent staining for fibrin and P-selectin. RESULTS: Injured mice that did not receive the P-selectin antibody showed a robust fourfold to fivefold increase in fibrin accumulation in both coup and contrecoup tissues (fluorescence per um of arterial wall) compared to uninjured sham mice. In contrast, mice pretreated with P-selectin blocking antibody showed no significant increase in fibrin accumulation on either side of the lungs after blunt thoracic trauma. No difference in mean fibrin deposition was found between sham controls that received the P-selectin-blocking antibody and those that received an isotype control antibody. CONCLUSION: P-selectin expression increases at the pulmonary arterial luminal surface following blunt thoracic trauma. In addition, P-selectin-blocking in vivo prevents pulmonary arterial fibrin accumulation after blunt thoracic trauma, confirming that P-selectin is necessary for de novo pulmonary arterial thrombosis after traumatic injury.

A cell-extrinsic ligand acquired by activated T cells in lymph node can bridge L-selectin and P-selectin.

P-selectin expressed on activated endothelia and platelets supports recruitment of leukocytes expressing P-selectin ligand to sites of inflammation. While monitoring P-selectin ligand expression on activated CD8+ T cells in murine adoptive transfer models, we observed two distinct ligands on responding donor cells, the canonical cell-intrinsic P-selectin ligand PSGL-1 and a second undocumented P-selectin ligand we provisionally named PSL2. PSL2 is unusual among selectin ligands in that it is cell-extrinsic, loaded onto L-selectin expressed by activated T cells but not L-selectin on resting naïve CD8+ T cells. PSL2 display is highest on activated T cells responding in peripheral lymph nodes and low on T cells responding in spleen suggesting that the original source of PSL2 is high endothelial venules, cells known to produce L-selectin ligands. PSL2 is a ligand for both P-selectin and L-selectin and can physically bridge the two selectins. The L-selectin/PSL2 complex can mediate P-selectin-dependent adherence of activated T cells to immobilized P-selectin or to activated platelets, either independently or cooperatively with PSGL-1. PSL2's capacity to bridge between L-selectin on activated T cells and P-selectin reveals an undocumented and unanticipated activity of cell-extrinsic selectin ligands in mediating selectin-selectin connectivity. The timing and circumstances of PSL2 detection on T cells, together with its capacity to support adherence to P-selectin-bearing substrates, are consistent with P-selectin engagement of both PSGL1 and the L-selectin/PSL2 complex during T cell recruitment. Engagement of PSGL-1 and L-selectin/PSL2 would likely deliver distinct signals known to be relevant in this process.

T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding.

Objective- Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. Approach and Results- We used wild-type mice and Selplg-/- and Fut7-/- mice defective in P-selectin binding and lymphopenic Rag2-/- mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4+ memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. Conclusions- The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke.

Rift valley fever viral load correlates with the human inflammatory response and coagulation pathway abnormalities in humans with hemorrhagic manifestations.

Rift Valley fever virus is an arbovirus that affects both livestock and humans throughout Africa and in the Middle East. Despite its endemicity throughout Africa, it is a rare event to identify an infected individual during the acute phase of the disease and an even rarer event to collect serial blood samples from the affected patient. Severely affected patients can present with hemorrhagic manifestations of disease. In this study we identified three Ugandan men with RVFV disease that was accompanied by hemorrhagic manifestations. Serial blood samples from these men were analyzed for a series of biomarkers specific for various aspects of human pathophysiology including inflammation, endothelial function and coagulopathy. There were significant differences between biomarker levels in controls and cases both early during the illness and after clearance of viremia. Positive correlation of viral load with markers of inflammation (IP-10, CRP, Eotaxin, MCP-2 and Granzyme B), markers of fibrinolysis (tPA and D-dimer), and markers of endothelial function (sICAM-1) were all noted. However, and perhaps most interesting given the fact that these individuals exhibited hemorrhagic manifestations of disease, was the finding of a negative correlation between viral load and P-selectin, ADAMTS13, and fibrinogen all of which are associated with coagulation pathways occurring on the endothelial surface.

Mouse Platelet Ral GTPases Control P-Selectin Surface Expression, Regulating Platelet-Leukocyte Interaction.

OBJECTIVE: RalA and RalB GTPases are important regulators of cell growth, cancer metastasis, and granule secretion. The purpose of this study was to determine the role of Ral GTPases in platelets with the use of platelet-specific gene-knockout mouse models. APPROACH AND RESULTS: This study shows that platelets from double knockout mice, in which both GTPases have been deleted, show markedly diminished (≈85% reduction) P-selectin translocation to the surface membrane, suggesting a critical role in α-granule secretion. Surprisingly, however, there were only minor effects on stimulated release of soluble α- and δ-granule content, with no alteration in granule count, morphology, or content. In addition, their expression was not essential for platelet aggregation or thrombus formation. However, absence of surface P-selectin caused a marked reduction (≈70%) in platelet-leukocyte interactions in blood from RalAB double knockout mice, suggesting a role for platelet Rals in platelet-mediated inflammation. CONCLUSIONS: Platelet Ral GTPases primarily control P-selectin surface expression, in turn regulating platelet-leukocyte interaction. Ral GTPases could therefore be important novel targets for the selective control of platelet-mediated immune cell recruitment and inflammatory disease.

Platelet-Derived MRP-14 Induces Monocyte Activation in Patients With Symptomatic Peripheral Artery Disease.

BACKGROUND: Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear. OBJECTIVES: The authors sought to investigate the role of platelets in a cohort of symptomatic PAD. METHODS: The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease. RESULTS: Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events. CONCLUSIONS: The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103).

In vivo multiplex molecular imaging of vascular inflammation using surface-enhanced Raman spectroscopy.

Vascular immune-inflammatory responses play a crucial role in the progression and outcome of atherosclerosis. The ability to assess localized inflammation through detection of specific vascular inflammatory biomarkers would significantly improve cardiovascular risk assessment and management; however, no multi-parameter molecular imaging technologies have been established to date. Here, we report the targeted in vivo imaging of multiple vascular biomarkers using antibody-functionalized nanoparticles and surface-enhanced Raman scattering (SERS). Methods: A series of antibody-functionalized gold nanoprobes (BFNP) were designed containing unique Raman signals in order to detect intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin using SERS. Results: SERS and BFNP were utilized to detect, discriminate and quantify ICAM-1, VCAM-1 and P-selectin in vitro on human endothelial cells and ex vivo in human coronary arteries. Ultimately, non-invasive multiplex imaging of adhesion molecules in a humanized mouse model was demonstrated in vivo following intravenous injection of the nanoprobes. Conclusion: This study demonstrates that multiplexed SERS-based molecular imaging can indicate the status of vascular inflammation in vivo and gives promise for SERS as a clinical imaging technique for cardiovascular disease in the future.

Platelets are activated in ANCA-associated vasculitis via thrombin-PARs pathway and can activate the alternative complement pathway.

BACKGROUND: In this study, we investigated the mechanism of platelet activation in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), as well as the activation of the alternative complement pathway by platelets in AAV. METHODS: CD62P and platelet-leukocyte aggregates in AAV patients were tested by flow cytometry. Platelets were stimulated by plasma from active AAV patients. The effect of the thrombin-protease-activated receptors (PARs) pathway was evaluated by blocking thrombin or PAR1 antagonists. After platelets were activated by plasma from AAV patients, Ca/Mg-Tyrode's buffer and Mg-EGTA buffer were used to measure complement activation in liquid phase and on the surface of platelets. RESULTS: The levels of CD62P-expressing platelets and platelet-leukocyte aggregates were significantly higher in active AAV patients than those in remission and normal controls. Platelets were activated by plasma from active AAV patients (percentage of CD62P-expressing platelets, 97.7 ± 3% vs. 1 ± 0.2%, p < 0.0001, compared with those incubated with healthy donor plasma), and this was inhibited by thrombin or PAR1 antagonists (percentage of CD62P-expressing platelets, 97.7 ± 3% vs. 2.7 ± 1%, p < 0.0001, 97.7 ± 3% vs. 5 ± 1.4%, p < 0.0001, respectively). Platelets activated by plasma from AAV patients could trigger complement activation via the alternative pathway, as demonstrated by significant elevation of C3a, C5a, and sC5b-9 and significantly more C3c and C5b-9 deposition on the surface of platelets. CONCLUSIONS: Platelets were activated in AAV patients, and such activation was at least partially attributed to the thrombin-PARs pathway. Activated platelets triggered the alternative complement pathway in AAV.

Immune Activation of Platelets in Response to Serial Phlebotomy in Pigtailed Macaques (Macaca nemestrina).

Serial phlebotomy is a common sampling practice for repeated-measures studies in biomedical research. In NHP, the effect of serial blood collection on RBC parameters has been characterized, but the effects on platelet parameters and other aspects of the hemogram have not been well studied. We sought to characterize the circulating platelet phenotype throughout the course of 7 serial phlebotomies spanning 30 d in pigtailed macaques (Macaca nemestrina). Phlebotomy was performed on 23 animals at days 0, 2, 4, 7, 10, 21, and 30 to quantify the circulating platelet count and markers of both hemostatic and immune platelet activation. Platelet immune activation was characterized by increases in surface MHC class I and II expression and increases in circulating platelet-leukocyte aggregates. These changes occurred in the absence of increases in the prohemostatic markers P-selectin and CD40L and without evidence of adverse clinical effects. Mild increases in platelet count, mean platelet volume, and immune activation occurred early in the study. After day 21, mean platelet volume and other hematologic parameters returned to baseline while changes in platelet count and immune activation were greater than during the first 10 d of the study. These data demonstrate that serial phlebotomy in NHP has delayed effects on platelet parameters, which may be a source of clinically silent, immunologic and physiologic variability within repeated measures studies. The impact of these effects on research aims should be considered when designing protocols requiring serial phlebotomy in NHP.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003.

BACKGROUND: Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. METHODS: Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). RESULTS: Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. CONCLUSIONS: Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.