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.     

Identification of dihydropteridine reductase in human platelets

Normal human platelets were shown to contain the enzyme dihydropteridine reductase. The enzyme was not found in a variety of other cells of hematogenous origin. Partial purification and kinetic and physical data indicated that the platelet enzyme is similar to that previously characterized from liver. Dihydropteridine reductase is important for the regeneration of tetrahydrobiopterin, a required cofactor in hydroxylation reactions involved in biogenic amine formation. The presence of the enzyme may indicate that some synthesis de novo of serotonin and/or catecholamines occurs in platelets, as opposed to a purely storage and transport function. In addition, screening for hyperphenylalaninemia due to dihydropteridine reductase deficiency may become feasible by assaying platelets for enzyme activity.     

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.     

The ratio of older women to men: historical perspectives and cross-national comparisons

In nearly all populations throughout the world there are substantially more older women than men. Although there are many biological explanations for why women have greater longevity than men, the higher proportion of women in the older population appears to be a phenomenon of the twentieth century. Using contemporary data on population size and life expectancy in a large number of countries and historical life table data from a diverse subset of countries, cross-national contrasts and historical trends in the female to male ratio are explored. In the 1990's, only 4 countries had fewer women than men in the age group 75 years and older. The number of women per 100 men aged 75+ in the remainder of the world's countries ranged from 100 to 355. In general, countries with a lower overall life expectancy had a lower number of women per 100 men aged 75+, while countries with higher overall life expectancy had a higher female to male ratio in this age group. A hundred years ago there were nearly equal numbers of women and men aged 75+ in many countries. In all countries studied, the female to male ratio increased as the century progressed. Historical life table data were used to calculate the probability of surviving through 5 stages of life: ages 0 to 5, 5 to 15, 15 to 40, 40 to 65, and 65 to 85. Although the probability of survival through all age intervals increased dramatically during the century, the current disparity in the size of the older populations of men and women can be explained primarily by the divergence in male and female probabilities of survival for the two older age intervals as the century progressed. Thus, with higher life expectancy, whether it be comparing countries or over time within a country, the proportion of the older population that is female is greater. Changes in survival probability in middle and late life, rather than childhood and young adulthood, have been responsible for the increased number of women compared to men in the older population.     

Predictive Value of Admission N-Terminal Pro-B-Type Natriuretic Peptide and Renal Function in Older People Hospitalized for Dyspnoea

Background. We investigated the relationship between NT-pro-BNP, glomerular filtration rate (GFR), and all-cause mortality rates in a cohort of older people discharged from an internal medicine unit after admission for dyspnoea. Patients and Methods. NT-pro-BNP was evaluated in serum samples of 134 patients aged 80 ± 6 years who presented to a single academic centre with worsening dyspnoea. History data and anthropometric, clinical, and biochemical parameters including GFR were collected at the time of admission. 119 out of 134 were discharged alive from hospital and were included in the follow-up of 779 ± 370 days. Results. 35 out of 119 subjects died after a follow-up of 266 ± 251 days. Cox proportional hazards model showed that GFR and Ln (NT-pro-BNP) were predictors for all-cause mortality with estimated hazard ratios of 0.969 (95% confidence interval: 0.950–0.988; P = 0.001) and 2.360 (95% confidence interval: 1.208–4.610; P = 0.012), respectively. Patients characterized by high NT-pro-BNP levels and GFR ≥ 60 mL/min/1.73 m² showed a dramatic reduction in survival duration compared with the groups with different combinations of the two variables (P = 0.008). Conclusions. In the elderly, NT-pro-BNP and GFR are predictors of all-cause mortality after admission because of dyspnoea. Since the fact that subjects with high NT-pro-BNP and GFR ≥ 60 mL/min/1.73 m² exhibited a reduced survival, high admission NT-pro-BNP suggests future negative outcome.     

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.