Packaging of supplemented urokinase into alpha granules of in vitro-grown megakaryocytes for targeted nascent clot lysis.

ABSTRACT: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

Studies of infused megakaryocytes into mice support a "catch-and-release" model of pulmonary-centric thrombopoiesis.

Many aspects of thrombopoiesis, the release of platelets from megakaryocytes (Mks), remain under debate, including where this process occurs. Murine lung in situ -microscopy studies suggested that a significant fraction of circulating platelets were released from lung-entrapped, marrow-derived Mks. We now confirm these in situ studies that endogenous mMks are entrapped in the lungs and show that intravenously infused in vitro -differentiated, mature murine (m) and human (h) Mks are similarly entrapped followed by shedding of their cytoplasm over ∼30 minutes with a peak number of released platelets occurring 1.5-4 hours later. However, while infused Mks from both species shed large intrapulmonary cytoplasmic fragments that underwent further processing into platelet-sized fragments, the two differed: many mMks escaped from and then recycled back to the lungs, while most hMks were enucleated upon first intrapulmonary passage. Infused immature hMks, inflammatory hMks, umbilical cord-blood-derived hMks and immortalized Mk progenitor cell (imMKCL)-derived hMks were also entrapped in the lung of recipient mice, and released their cytoplasm, but did so to different degrees. Intraarterial infused hMks resulted in few Mks being entrapped in tissues other than the lungs and was accompanied by a blunted and delayed rise in circulating human platelets. These studies demonstrate that the lung entraps and processes both circulating Mks and released large cytoplasmic fragments consistent with a recent lung/heart murine study and support a pulmonary-centric "catch-and-release" model of thrombopoiesis. Thus, thrombopoiesis is a drawn-out process with the majority of cytoplasmic processing derived from Mks occurring in the pulmonary bed. Key Points: Infused in vitro -differentiated megakaryocytes synchronously release cytoplasmic fragments highly selectively in the pulmonary bed. Large, released megakaryocyte fragments recycle to the lungs, undergo further fission, terminally form platelets.

Packaging of supplemented urokinase into naked alpha-granules of in vitro -grown megakaryocytes for targeted therapeutic delivery.

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34 + hematopoietic cell-derived megakaryocytes. CD34 + -megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34 + -\megakaryocytes was mediated in part via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWF R1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWF R1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules from in vitro- generated megakaryocytes. Key points: Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbß3 receptors and is functionally available from activated platelets.

A de novo proximal 6q deletion confirmed by array comparative genomic hybridization.

Deletions of chromosome 6q, particularly in the proximal region, are relatively rare. Here, we report on a de novo interstitial deletion of (6)(q13q16.2) in a girl with facial dysmorphism, congenital hip dislocation, porencephaly, and brain atrophy. Array comparative genomic hybridization analysis showed arr 6q13q16.2(73,378,824?99,824,130), demonstrating higher resolution than the conventional cytogenetic findings, del(6)(q12q15). The clinical data were analyzed and compared with those of similar patients previously reported in the literature.

The clinical significance of food specific IgE/IgG4 in food specific atopic dermatitis.

Food is closely associated with the pathogenesis of atopic dermatitis. Food allergy is usually mediated by IgE antibody to specific food proteins and determination of specific IgE antibody is the basis of the common diagnostic test for food allergy. IgG4 have been reported as blocking antibody and the protective effects of blocking antibody may be clear in inhalant allergy. However, the role of IgG4 in food allergy is still a matter of debate. In this study, the clinical significance of food allergen-specific IgE/IgG4 in atopic dermatitis was investigated and compared with that of IgE. A total of 97 patients who fulfilled the diagnostic criteria for atopic dermatitis participated in this study. Skin prick test and allergy patch test were performed. Specific IgE and IgG4 concentration were measured using allergy protein chip, 'AllergyChip'. Double blinded placebo controlled food challenge test (DBPCFC) was performed for the diagnosis of allergy to milk, egg white, wheat, and soybean. DBPCFCs for milk, egg white, soybean, and wheat were performed. The positive rates were 31.7% (19/60) in milk, 36.7% (18/49) in egg white, 30.4% (7/23) in soybean, and 34.8% (8/23) in wheat. Mean IgE/IgG4 levels in DBPCFC (+) subjects is higher than those in DBPCFC (-) subjects in all food items studied. Of them, there were significantly different between two groups in egg white and wheat (Egg white in DBPCFC (+) vs. (-): 0.4 +/- 0.3 vs. 0.2 +/- 0.2, wheat in DBPCFC (+) vs. (-): 1.2 +/- 1.2 vs. 0.3 +/- 0.3, p < 0.05). Allergen-specific IgE/IgG4 may provide one of the clues to understand the mechanism of food allergy in atopic dermatitis. The present study suggests that protein microarray can be one of the useful methods to assess ongoing status of allergic diseases.