ALBERT

Description: Allograft transplantation requires chronic immunosuppression, but there is no effective strategy to evaluate the long-term maintenance of immunosuppression other than assessment of graft function. The ability to monitor naive alloreactive T cells would provide an alternative guide for drug therapy at early, preclinical stages of graft rejection and for evaluating tolerance-inducing protocols. To detect and quantify naive alloreactive T cells directly ex vivo, we used the unique ability of naive T cells to rapidly produce TNF-α but not IFN-γ. Naive alloreactive T cells were identified by the production of TNF-α after a 5-hour in vitro stimulation with alloantigen and were distinguished from effector/memory alloreactive T cells by the inability to produce IFN-γ. Moreover, naive alloreactive T cells were not detected in mice tolerized against specific alloantigens. The frequency of TNF-α–producing cells was predictive for rejection in an in vivo cytotoxicity assay and correlated with skin allograft rejection. Naive alloreactive T cells were also detected in humans, suggesting clinical relevance. We conclude that rapid production of TNF-α can be used to quantify naive alloreactive T cells, that it is abrogated after the induction of tolerance, and that it is a potential tool to predict allograft rejection.

Description: Systemic mastocytosis (SM) is a clonal myeloproliferative disease with variable clinical manifestations. The D816V mutation in the c-kit gene, present in over 90% of adult patients with SM, results in constitutive activation of the receptor tyrosine kinase and is believed to be related to disease pathogenesis. Although the majority of patients with SM lack peripheral blood eosinophilia, a subgroup exists and is classified as SM with eosinophilia (SM-eo). Recently, several reports of patients with SM-eo described the presence of either the Kit D816V mutation or the FIP1L1/PDGFRA fusion oncogene. Numerous similarities between patients with FIP1L1/PDGFRA and KIT D816V-associated peripheral blood eosinophilia have caused confusion about the management and specifically the role of imatinib in the treatment of these patients. It is of paramount importance to distinguish these two groups with pathologically similar, but molecularly and clinically distinct diseases. We thus compared the clinical, laboratory, and molecular features of 12 patients who met WHO criteria for SM (including presence of the D816V kit mutation) and had associated peripheral eosinophilia with those of 17 patients with peripheral eosinophilia and the FIP1L1/PDGFRA fusion oncogene (diagnosed with HES and evaluated at the same institution) and to the published reports of FIP1L1/PDGFRA-HES patients. Based on these comparisons, a number of clinical features appeared to be of potential use in distinguishing these two disorders. The presence of cardiac symptoms, a total serum tryptase under 60 ng/ml or the presence of either scattered mast cells or loose aggregates was found to be suggestive of FIP1L1/PDGFRA-associated disease. The presence of urticaria pigmentosa, a total serum tryptase over 150 ng/ml, the presence of dense mast cell aggregates and female sex were suggestive of Kit D816V-associated disease. To confirm and standardize this clinical classification, statistical methods were employed to test 21 possible risk factors for their ability to distinguish Kit and FIP1L1/PDGFRA-associated disease. Calculated risk factor scores were developed based on this analysis. Applying this risk factor based system, 16/17 FIP1L1/PDGFRA patients were classified correctly, with one patient neutral and all 12 Kit D816V SM-eo patients were classified correctly. Thirty four FIP1L1/PDGFRA patients in the literature were available for analysis, although all risk factors to create the score were not available for all patients. Despite this, 25/34 FIP1L1/PDGFRA patients were correctly predicted as FIP1L1/PDGFRA, 4/34 patients were neutral and 5/34 were misclassified as Kit D816V-associated SM-eo. These data suggest that the risk factor-based system presented in this study is useful in distinguishing imatinib-sensitive FIP1L1/PDGFRA-associated disease from imatinib-resistant Kit D816V-associated disease. Parameter SM-eo FIP1L1/PDGFRA HES Number patients 12 17 Male/Female 7/5 17/0 Cardiac symptoms 0/12 6/15 UP 7/12 0/15 Mean serum tryptase (ng/ml) 229 28 (n=13) Dense marrow aggregates 12/12 1/10

Description: Based on previous studies showing the efficacy of FactorIXa (FIXa) blockade using an active site-blocked form of this coagulation enzyme, we speculated that partial inhibition of the intrinsic coagulation pathway would offer a novel approach to attenuate intravascular clot formation without promoting untoward bleeding. Here we describe the anticoagulant activity of TTP889, a small molecule partial inhibitor of FIXa activity. TTP889 is orally absorbed with a PK profile that is conducive to once daily dosing. It is selective for FIXa in that it shows little to no activity against several other proteases in the clotting cascade including FXa, FXIa, FXIIa or FVIIa in a unique clotting assay. In vivo, TTP889 inhibited fibrin deposition in a rat arteriovenous (A/V) shunt model. In this model, vehicle treated rats had 104mg ± 43 of fibrin deposited on a silk thread after a 15 minute shunt while TTP889 treated rats had significantly less fibrin deposited (39mg ± 18 p=

Description: Human mast cells are found in skin and mucosal surfaces and next to blood vessels. They play a sentinel cell role in immunity, recognizing invading pathogens and producing proinflammatory mediators. Mast cells can recruit granulocytes, and monocytes in allergic disease and bacterial infection, but their ability to recruit antiviral effector cells such as natural killer (NK) cells and T cells has not been fully elucidated. To investigate the role of human mast cells in response to virus-associated stimuli, human cord blood–derived mast cells (CBMCs) were stimulated with polyinosinic·polycytidylic acid, a double-stranded RNA analog, or infected with the double-stranded RNA virus, reovirus serotype 3 Dearing for 24 hours. CBMCs responded to stimulation with polyinosinic·polycytidylic acid by producing a distinct chemokine profile, including CCL4, CXCL8, and CXCL10. CBMCs produced significant amounts of CXCL8 in response to low levels of reovirus infection, while both skin- and lung-derived fibroblasts were unresponsive unless higher doses of reovirus were used. Supernatants from CBMCs infected with reovirus induced substantial NK cell chemotaxis that was highly dependent on CXCL8 and CXCR1. These results suggest a novel role for mast cells in the recruitment of human NK cells to sites of early viral infection via CXCL8.

Description: Background: Peripheral Blood Progenitor Cells (PBPC) cryopreserved in DMSO are thawed at the patient’s bedside and infused immediately. Usually the infusions are completed without incident. There are occasions when mild/severe transfusion related reactions occur. When this does occur, the infusion is either slowed or paused for a short period of time. When reactions are severe enough to warrant stoppage of the infusion, transplant facilities maybe faced with discarding any remaining thawed cells. We have explored the question of salvaging the remaining cells by re-cryopreservation using two cryopreservation methods. Study Design: Twenty PBPC were thawed in a 37C water bath until the internal temperature of the cells was approx 2-6C. The PBPC were not washed and allowed to remain at room temperature for 15 minutes. Ten PBPC were then placed on ice for 45 minutes. This simulated the transfer of cells to an off-site cryopreservation lab. These cells were then transferred to a new cryo bag, no change in the cryoprotectant solutions, and placed into a rate controlled freezer (RCF) bringing their temperature back to

Description: Although imatinib is clearly the treatment of choice for FIP1L1/PDGFRA-positive chronic eosinophilic leukemia (CEL), little is known about optimal dosing, duration of treatment, and the possibility of cure in this disorder. To address these questions, 5 patients with FIP1L1/PDGFRA-positive CEL with documented clinical, hematologic, and molecular remission on imatinib (400 mg daily) and without evidence of cardiac involvement were enrolled in a dose de-escalation trial. The imatinib dose was tapered slowly with close follow-up for evidence of clinical, hematologic, and molecular relapse. Two patients with endomyocardial fibrosis were maintained on imatinib 300 to 400 mg daily and served as controls. All 5 patients who underwent dose de-escalation, but neither of the control patients, experienced molecular relapse (P 〈 .05). None developed recurrent symptoms, and eosinophil counts, serum B12, and tryptase levels remained suppressed. Reinitiation of therapy at the prior effective dose led to molecular remission in all 5 patients, although 2 patients subsequently required increased dosing to maintain remission. These data are consistent with suppression rather than elimination of the clonal population in FIP1L1/PDGFRA-positive CEL and suggest that molecular monitoring may be the most useful method in determining optimal dosing without the risk of disease exacerbation. This trial was registered at http://www.clinicaltrials.gov as no. NCT00044304.

Description: Several compounds currently in development for the treatment of thrombotic disorders demonstrate high levels of specificity for single targets of the blood coagulation cascade such as factor Xa and thrombin. However, development of a single molecule dual inhibitor against factor Xa and thrombin may expand the efficacy to safety ratio of treatment options for arterial and venous thrombosis. The objective of this study was to determine if simultaneous administration of PD 0313052, a selective Xa inhibitor and argatroban, a direct thrombin inhibitor, would lead to a synergistic antithrombotic effect in a rabbit AV shunt model of thrombosis. Intravenous administration of PD 0313052 alone at doses of 0.1, 0.3, and 1.0 mg/kg/min resulted in thrombus weight (TW) reductions of 11±3, 25±10 and 67±7 % compared to the vehicle group. Argatroban at 1, 3 and 10 mg/kg/min reduced TW 16±13, 47±10 and 75±6 %. When PD 0313052 was administered at 0.1 mg/kg/min in combination with argatroban at 1, 3 or 10 mg/kg/min TW was reduced 50±7, 60±7 and 82±9 %. Likewise, argatroban at 1 mg/kg/min combined with 0.1, 0.3 or 1mg/kg/min of PD 0313052 resulted in TW reductions of 56±9, 60±9 and 84±5 %, respectively. At the lowest combined doses of PD 0313052 and argatroban there was no change in bleeding time relative to the additive fold-increases from each drug alone. The EC50 of intravenously administered PD 0313052 and argatroban was 67±23 and 178±58 ng/ml, respectively. When the drugs were combined the EC50 was reduced to 12±6 ng/ml with the PD 0313052/argatroban combination and to 83±29 ng/ml with the argatroban/PD 0313052 combination. A synergistic effect was also observed in an ex vivo assay of thrombin generation (TG). Predicted additive inhibition of TG based on the individual effects of each compound was −9±7, 9±2 and 29±7 % compared to 10±5, 32±5 and 55±3 % with the 313052/argatroban combination. The predicted effects of the argatroban/PD 0313052 combination was −9±7, 1±7 and 16±9 % compared to the actual inhibition of 5±3, 14±5 and 31±7 %. These results demonstrate a significant synergistic antithrombotic effect by combining low doses of a factor Xa and a thrombin inhibitor and support the hypothesis that development of a single molecule inhibitor against different hemostatic targets may offer greater efficacy in the prevention and treatment of venous and arterial thrombosis.

Description: The purpose of this study was to characterize a large group of infants with complete DiGeorge anomaly and to evaluate the ability of thymus transplantation to reconstitute immune function in these infants. DiGeorge anomaly is characterized by varying defects of the heart, thymus, and parathyroid glands. Complete DiGeorge anomaly refers to the subgroup that is athymic (〈 1%). The characteristics of 54 subjects at presentation and results from 44 consecutive thymus transplantations are reported. Remarkably, only 52% had 22q11 hemizygosity and only 57% had congenital heart disease requiring surgery. Thirty-one percent developed an atypical phenotype with rash and lymphadenopathy. To date, 33 of 44 subjects who received a transplant survive (75%) with post-transplantation follow-up as long as 13 years. All deaths occurred within 12 months of transplantation. All 25 subjects who were tested 1 year after transplantation had developed polyclonal T-cell repertoires and proliferative responses to mitogens. Adverse events developing after transplantation included hypothyroidism in 5 subjects and enteritis in 1 subject. In summary, diagnosis of complete DiGeorge anomaly is challenging because of the variability of presentation. Thymus transplantation was well tolerated and resulted in stable immunoreconstitution in these infants.

Description: Hemostatic parameters of fawn-hooded (FH) rats with an inherited hemorrhagic diathesis were compared with those of normal rats. Prothrombin time, partial thromboplastin time, plasma factor VIII, plasma fibrinogen, and platelet count and volume were normal. Bleeding time (BT) in FH rats was over 15 min vs. 1-8 min in controls, and platelet retention in glass-bead columns was reduced. Transfusion of platelet concentrates from normal rats corrected the BT of thrombocytopenic FH rats, but FH rat platelets did not shorten the BT of thrombocytopenic controls. ADP-induced aggregation, measured turbidimetrically in heparinized FH platelet-rich plasma, was normal. Connective tissue did not aggregate platelets of FH rats and released a subnormal amount of 14C-serotonin. In comparison with control platelets washed FH platelets aggregated by thrombin released virtually no ATP and ADP. Platelets, 10,11 of normal rats contained 4.49 µmoles ATP, 0.98 µmoles ADP, and 0.88 µmoles serotonin compared with only about one-half as much ATP and ADP and one-third as much serotonin in platelets of FH rats. These low values suggest the absence of the platelet, "release pool" rather than a defective release mechanism. The hemorrhagic diathesis seen in the FH rats resembles the platelet defect with storage pool deficiency observed in man.2

Description: Patent lymphatic filariasis is characterized by antigen-specific T-cell unresponsiveness with diminished IFN-γ and IL-2 production and defects in dendritic cell (DC) function. Because Toll-like receptors (TLRs) play an important role in pathogen recognition and TLR expression is diminished on B and T cells of filaria-infected individuals, we examined the effect of live microfilariae (mf) on expression and function of TLRs in human DCs. We show that mf-exposed monocyte-derived human DCs (mhDCs) demonstrate marked diminution of TLR3 and TLR4 mRNA expression compared with mf-unexposed mhDCs that translated into loss of function in response to appropriate TLR ligands. Exposure to mf significantly down-regulated production of IFN-α, MIP-1α, IL-12p70, and IL-1α following activation with poly I:C, and of IL-12p40 following activation with poly I:C or LPS. mRNA expression of MyD88, the adaptor molecule involved in TLR4 signaling, was significantly diminished in mhDCs after exposure to mf. Moreover, mf interfered with NF-κB activation (particularly p65 and p50) following stimulation with poly I:C or LPS. These data suggest that mf interfere with mhDC function by altering TLR expression and interfering with both MyD88-dependent signaling and a pathway that ultimately diminishes NF-κB activity. This down-regulated NF-κB activity impairs mhDC-produced cytokines needed for full T-cell activation.