Signal transduction by the platelet Fc receptor

We have evaluated the mechanism by which crosslinking human platelet Fc receptor (FcR) for IgG triggers platelet aggregation and the platelet release reaction. Platelet FcR was crosslinked by incubating purified human platelets with anti-FcRII monoclonal antibody and F(ab')2 anti- mouse Ig. The resultant [Ca2+]i increase, monitored by Fura-2 and measured in the absence of extracellular Ca2+, reached a peak of 750 +/- 50 nmol/L. The effects of cyclooxygenase inhibitors, aspirin and indomethacin, and a phospholipase A2 inhibitor, dibromoacetophenone, were examined. Regardless of the inhibitor, at least 25% of the [Ca2+]i increase remained. Thrombin (0.2 U/mL) stimulated an immediate [Ca2+]i increase that reached 1.95 +/- 0.8 mumol/L. The [Ca2+]i increase generated by thrombin was only slightly reduced by these inhibitors. Crosslinking the FcRII of platelets resulted in a fivefold increase in the production of [3H]inositol phosphates, (IP) which, in the absence of extracellular Ca2+ was insensitive to aspirin. The activation of a [Ca2+]i increase along with the measured increases in IP indicate that FcRII crosslinking leads to the activation of phospholipase C (PLC). In contrast to thrombin, platelet activation via FcRII depends to a large extent on arachidonic acid metabolites. However, neither cyclooxygenase nor phospholipase A2 inhibitors completely blocked FcRII-stimulated [Ca2+]i increase. These observations led us to propose that crosslinking of platelet FcRII initially activates PLC.     

Preclinical to Clinical Translation of CNS Transporter Occupancy of TD-9855, a Novel Norepinephrine and Serotonin Reuptake Inhibitor

Background:

Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters.

Methods:

We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor.

Results:

TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC₅₀ of 11.7ng/mL and 50.8ng/mL, respectively, consistent with modest selectivity for NET in vivo.Accounting for species differences in protein binding, the projected human NET and SERT plasma EC₅₀ values were 5.5ng/mL and 23.9ng/mL, respectively. A single-dose, open-label PET study (4–20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [¹¹C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [¹¹C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30–40h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC₅₀ for NET was estimated to be 1.21ng/mL, and at doses of greater than 4mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35ng/mL.

Conclusions:

These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation.     

High-dose etoposide and cyclophosphamide without bone marrow transplantation for resistant hematologic malignancy

Seventy-five patients with resistant acute leukemia or lymphoma received high-dose cyclophosphamide and etoposide to explore the activity of this combination in resistant hematologic malignancies, and to determine the maximum doses of these drugs that can be combined without bone marrow transplantation. Etoposide was administered over 29 to 69 hours by continuous infusion corresponding to total doses of 1.8 g/m2 to 4.8 g/m2. Cyclophosphamide, 50 mg/kg/d, was administered on 3 or 4 consecutive days total 150 to 200 mg/kg ideal body weight). At all dose levels myelosuppression was severe but reversible. Mucosal toxicity was dose-limiting with the maximum tolerated dose level combining etoposide 4.2 g/m2 with cyclophosphamide 200 mg/kg. Continuous etoposide infusion produced stable plasma levels that were lower than would be achieved after administration by short intravenous infusion, and this could explain our ability to escalate etoposide above the previously reported maximum tolerated dose. There were 28 complete (35%) and 12 partial (16%) responses. Median duration of complete response (CR) was 3.5 months (range 1.1 to 20+). Seventeen of 40 patients (42%) with acute myelogenous leukemia (AML) achieved CR, including 6 of 20 (30%) with high-dose cytosine arabinoside resistance. We conclude that bone marrow transplantation is not required after maximum tolerated doses of etoposide and cyclophosphamide. This regimen is active in resistant hematologic neoplasms, and the occurrence of CR in patients with high-dose cytosine arabinoside-resistant AML indicates a lack of complete cross-resistance between these regimens.     

Identification of an oncogenic form of the thrombopoietin receptor MPL using retrovirus-mediated gene transfer

Thrombopoietin and its receptor (MPL) are important regulators of megakaryopoiesis. We have identified an activating mutation of MPL using a combination of a retrovirus-mediated gene transfer and polymerase chain reaction-driven random mutagenesis. This point mutation causes a single amino acid substitution from Ser498 to Asn498 in the transmembrane region and abrogates factor-dependency of all interleukin-3-dependent cell lines tested. Murine interleukin-3- dependent Ba/F3 cells expressing the mutated but not the normal form of MPL were tumorigenic when transduced into syngeneic mice. Analysis of intracellular signaling pathways indicated that the mutant MPL protein constitutively activated two distinct signaling pathways, SHC-Raf-MAPK and JAK2-STAT3/STAT5.     

Secondary prevention of cryptogenic stroke in patients with patent foramen ovale: a systematic review and meta-analysis

The aim of our study is to compare patent foramen ovale (PFO) closure versus medical treatment and antiplatelet versus anticoagulant therapy in patients with cryptogenic stroke (CS) and PFO. We conducted a systematic review and meta-analysis with trial sequential analysis (TSA) of randomized trials. Primary outcomes are stroke or transient ischemic attack (TIA) and all-cause mortality. Secondary outcomes are peripheral embolism, bleeding, serious adverse events, myocardial infarction and atrial dysrhythmias. We performed an intention to treat meta-analysis with a random-effects model. We include six trials (3677 patients, mean age 47.3 years, 55.8% men). PFO closure is associated with a lower recurrence of stroke or TIA at a mean follow-up of 3.88 years compared to medical therapy [risk ratio (RR) 0.55, 95% CI 0.38–0.81; I²?=?40%]. The TSA confirms this result. No difference is found in mortality (RR 0.74, 95% CI 0.35–1.60; I²?=?0%), while PFO closure is associated with a higher incidence of atrial dysrhythmias (RR 4.55, 95% CI 2.16–9.60; I²?=?25%). The rate of the other outcomes is not different among the two groups. The comparison between anticoagulant and antiplatelet therapy shows no difference in terms of stroke recurrence, mortality and bleeding. There is conclusive evidence that PFO closure reduces the recurrence of stroke or TIA in patients younger than 60 years of age with CS. More data are warranted to assess the consequences of the increase in atrial dysrhythmias and the advantage of PFO closure over anticoagulants.     

Management of bisphosphonate‐related osteonecrosis of the jaw: a literature review

Osteonecrosis of the jaw (ONJ) is a serious side effect of bisphosphonate use in patients with osteoporosis, Paget's disease, hypercalcemia of malignancy, metastatic bone disease and multiple myeloma, although recently this complication has also been reported in patients under non‐bisphosphonate medication, such as denosumab and bevacizumab. The occurrence of ONJ is higher in oncology patients treated with high‐dose iv bisphosphonates than in osteoporosis patients treated with oral bisphosphonates. Although multiple hypotheses have been proposed, the exact pathogenic mechanism of ONJ still remains unclear. As treatment protocols based on randomized controlled trials (RCTs) do not exist, we critically reviewed the existing data concerning the management of bisphosphonate‐related osteonecrosis of the jaw, including the most recent data for the use of teriparatide and hyperbaric oxygen.     

Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony- forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.     

From primary progressive aphasia to corticobasal syndrome: two clinical and rCBF functional reports

We describe two cases, both presenting with a 2-year history of isolated language disorders, one compatible with logopenic variant and the other with non-fluent variant of primary progressive aphasia (PPA). Afterwards, each developed a corticobasal syndrome (CBS) with alien limb phenomenon and a multi-domain cognitive impairment. Regional cerebral perfusion (rCBF) study using 99mTc-ECD single photon emission computed tomography (SPECT) revealed hypoperfusion patterns consistent with these aphasia types and with the presence of limb apraxia. We report two cases of PPA variants associated with CBS and we suggest that SPECT rCBF correlates can be useful in making a differential diagnosis within the PPA spectrum.