Expression of specific granule markers on the cell surface of neutrophil cytoplasts

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6- fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12- binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.     

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.     

Red cell membrane stiffness in iron deficiency

The purpose of this study was to characterize red blood cell (RBC) deformability by iron deficiency. We measured RBC deformability to ektacytometry, a laser diffraction method for determining the elongation of suspended red cells subjected to shear stress. Isotonic deformability of RBC from iron-deficient human subjects was consistently and significantly lower than that of normal controls. In groups of rats with severe and moderate dietary iron deficiency, RBC deformability was also reduced in proportion to the severity of iron deficiency. At any given shear stress value, deformability of resealed RBC ghosts from both iron-deficient humans and rats was lower than that of control ghosts. However, increase of applied shear stress resulted in progressive increase in ghost deformation, indicating that ghost deformability was primarily limited by membrane stiffness rather than by reduced surface area-to-volume ratio. This was consistent with the finding that iron-deficient cells had a normal membrane surface area. In addition, the reduced mean corpuscular hemoglobin concentration (MCHC) and buoyant density of the iron-deficient rat cells indicated that a high hemoglobin concentration was not responsible for impaired whole cell deformability. Biochemical studies of rat RBC showed increased membrane lipid and protein crosslinking and reduced intracellular cation content, findings that are consistent with in vivo peroxidative damage. RBC from iron-deficient rats incubated in vitro with hydrogen peroxide showed increased generation of malonyldialdehyde, an end-product of lipid peroxidation, compared to control RBC. Taken together, these findings suggest that peroxidation could contribute in part to increased membrane stiffness in iron- deficient RBC. This reduced membrane deformability may in turn contribute to impaired red cell survival in iron deficiency.     

Subungual squamous cell carcinoma presenting with minimal nail changes: A factor in delayed diagnosis?

Squamous cell carcinoma of the nail bed is a relatively uncommon tumour that may be diagnosed only after considerable delay. The first case presented is a 79-year-old man with a history of discomfort and discoloration affecting the right thumbnail of 3 years duration. The second case is a 70-year-old man who presented with a recurrent, offensive discharge from beneath the left thumbnail of 40 years duration. Clinical examination of the affected digits revealed minor nail abnormalities. The presence of tumour was fully apparent only after removal of the nail plate and inspection and biopsy of the nail bed. The cases demonstrate that subungual squamous cell carcinoma may present with prolonged symptoms and a deceptively benign appearance. The importance of consideration of the possibility of malignancy, removal of the nail plate for inspection of the nail bed and appropriate biopsy is emphasized.