Mental health, pain and likelihood of opioid misuse among adults with sickle cell disease.

Depressive symptoms are prevalent in individuals living with sickle cell disease (SCD) and may exacerbate pain. This study examines whether higher depressive symptoms are associated with pain outcomes, pain catastrophizing, interference and potential opioid misuse in a large cohort of adults with SCD. The study utilized baseline data from the 'CaRISMA' trial, which involved 357 SCD adults with chronic pain. Baseline assessments included pain intensity, daily mood, the Patient Health Questionnaire (PHQ), the Generalized Anxiety Disorders scale, PROMIS Pain Interference, Pain Catastrophizing Scale, the Adult Sickle Cell Quality of Life Measurement Information System and the Current Opioid Misuse Measure. Participants were categorized into 'high' or 'low' depression groups based on PHQ scores. Higher depressive symptoms were significantly associated with increased daily pain intensity, negative daily mood, higher pain interference and catastrophizing, poorer quality of life and a higher likelihood of opioid misuse (all p < 0.01). SCD patients with more severe depressive symptoms experienced poorer pain outcomes, lower quality of life and increased risk of opioid misuse. Longitudinal data from this trial will determine whether addressing depressive symptoms may potentially reduce pain frequency and severity in SCD.

Protein kinase D1 conditional null mice show minimal bone loss following ovariectomy.

We previously found that 3- and 6-month-old male mice with conditional ablation of protein kinase D1 (PRKD1) in osteoprogenitor cells (expressing Osterix) exhibited reduced bone mass. Others have demonstrated similar effects in young female PRKD1-deficient mice. Here we examined the bone resorptive response of adult female floxed control and conditional knockout (cKO) mice undergoing sham surgery or ovariectomy (OVX). Femoral and tibial bone mineral density (BMD) values were significantly reduced upon OVX in control, but not cKO, females compared to the respective sham-operated mice. Micro-CT analysis showed that OVX significantly increased trabecular number and decreased trabecular spacing in cKO but not control mice. Finally, in control mice serum levels of a marker of bone resorption (pyridinoline crosslinks) and the osteoclast activator RANKL significantly increased upon OVX; however, no such OVX-induced increase was observed in cKO mice. Our results suggest the potential importance of PRKD1 in response to estrogen loss in bone.

Deletion of protein kinase D1 in osteoprogenitor cells results in decreased osteogenesis in vitro and reduced bone mineral density in vivo.

Protein kinase D1 (PRKD1) is thought to play a role in a number of cellular functions, including proliferation and differentiation. We hypothesized that PRKD1 in bone marrow-derived mesenchymal stem cells (BMMSC) could modulate osteogenesis. In BMMSCs from floxed PRKD1 mice, PRKD1 ablation with adenovirus-mediated Cre-recombinase expression inhibited BMMSC differentiation in vitro. In 3- and 6-month-old conditional knockout mice (cKO), in which PRKD1 was ablated in osteoprogenitor cells by osterix promoter-driven Cre-recombinase, bone mineral density (BMD) was significantly reduced compared with floxed control littermates. Microcomputed tomography analysis also demonstrated a decrease in trabecular thickness and bone volume fraction in cKO mice at these ages. Dynamic bone histomorphometry suggested a mineralization defect in the cKO mice. However, by 9 months of age, the bone appeared to compensate for the lack of PRKD1, and BMD was not different. Taken together, these results suggest a potentially important role for PRKD1 in bone formation.

Possible Role of Phosphatidylglycerol-Activated Protein Kinase C-ßII in Keratinocyte Differentiation.

BACKGROUND: The epidermis is a continuously regenerating tissue maintained by a balance between proliferation and differentiation, with imbalances resulting in skin disease. We have previously found that in mouse keratinocytes, the lipid-metabolizing enzyme phospholipase D2 (PLD2) is associated with the aquaglyceroporin, aquaporin 3 (AQP3), an efficient transporter of glycerol. Our results also show that the functional interaction of AQP3 and PLD2 results in increased levels of phosphatidylglycerol (PG) in response to an elevated extracellular calcium level, which triggers keratinocyte differentiation. Indeed, we showed that directly applying PG can promote keratinocyte differentiation. OBJECTIVE: We hypothesized that the differentiative effects of this PLD2/AQP3/PG signaling cascade, in which AQP3 mediates the transport of glycerol into keratinocytes followed by its PLD2-catalyzed conversion to PG, are mediated by protein kinase CßII (PKCßII), which contains a PG-binding domain in its carboxy-terminus. Method: To test this hypothesis we used quantitative RT-PCR, western blotting and immunocytochemistry. RESULTS: We first verified the presence of PKCßII mRNA and protein in mouse keratinocytes. Next, we found that autophosphorylated (activated) PKCßII was redistributed upon treatment of keratinocytes with PG. In the unstimulated state phosphoPKCßII was found in the cytosol and perinuclear area; treatment with PG resulted in enhanced phosphoPKCßII localization in the perinuclear area. PG also induced translocation of phosphoPKCßII to the plasma membrane. In addition, we observed that overexpression of PKCßII enhanced calcium- and PG-induced keratinocyte differentiation without affecting calcium-inhibited keratinocyte proliferation. CONCLUSION: These results suggest that the PG produced by the PLD2/AQP3 signaling module may function by activating PKCßII.

Preparation of primary cultures of mouse epidermal keratinocytes and the measurement of phospholipase D activity.

In this chapter information is provided about the outer layer of the skin, the epidermis, and the predominant cells comprising this epithelium, the keratinocytes. The evidence supporting a possible role for the lipid-metabolizing enzyme phospholipase D in regulating keratinocyte differentiation is also discussed. A detailed protocol for the preparation of primary cultures of epidermal keratinocytes from neonatal mice is described, to allow other investigators to obtain data concerning these important cells involved in forming and maintaining the mechanical and water permeability of the skin. Finally, a complete protocol for monitoring phospholipase D activity in intact cells is supplied in the hope that additional research will result in a better understanding of the role of phospholipase D in controlling keratinocyte proliferation and differentiation.

Aromatic amino acid activation of signaling pathways in bone marrow mesenchymal stem cells depends on oxygen tension.

The physiologic oxygen pressures inside the bone marrow environment are much lower than what is present in the peripheral circulation, ranging from 1-7%, compared to values as high as 10-13% in the arteries, lungs and liver. Thus, experiments done with bone marrow mesenchymal stem cells (BMMSCs) using standard culture conditions may not accurately reflect the true hypoxic bone marrow microenvironment. However, since aging is associated with an increased generation of reactive oxygen species, experiments done under 21%O2 conditions may actually more closely resemble that of the aging bone marrow environment. Aromatic amino acids are known to be natural anti-oxidants. We have previously reported that aromatic amino acids are potent agonists for stimulating increases in intracellular calcium and phospho-c-Raf and in promoting BMMSC differentiation down the osteogenic pathway. Our previous experiments were performed under normoxic conditions. Thus, we next decided to compare a normoxic (21% O2) vs. a hypoxic environment (3% O2) alone or after treatment with aromatic amino acids. Reverse-phase protein arrays showed that 3% O2 itself up-regulated proliferative pathways. Aromatic amino acids had no additional effect on signaling pathways under these conditions. However, under 21%O2 conditions, aromatic amino acids could now significantly increase these proliferative pathways over this "normoxic" baseline. Pharmacologic studies are consistent with the aromatic amino acids activating the extracellular calcium-sensing receptor. The effects of aromatic amino acids on BMMSC function in the 21% O2 environment is consistent with a potential role for these amino acids in an aging environment as functional anti oxidants.

Two new hemoglobin variants: Hb Tallahassee [α3(A1)Ser→Tyr; HBA2: c.11C>A] and Hb madison-NC [ß119(GH2)Gly→Ser; HBB: c.358G>A].

Of the 1570 reported hemoglobin (Hb) variants detected to date, 390 are α2-globin chain (some variants have yet to be identified by DNA analyses and are therefore presumed) and 827 are the result of mutations of the ß-globin chain. Due to their location on the Hb structure, only a minority of these variants result in a clinical phenotype; most are silent and are detected during routine surveillance, are found incidentally during other disease-related investigations or following newborn screening programs. In this report we discuss phenotype/genotype and molecular characteristics of two new Hb variants, both of which were clinically silent. One is an α2-globin chain variant located at codon 3 [α3(A1)Ser→Tyr; HBA2: c.11C > A] named Hb Tallahassee and the other is a ß-globin chain variant located at codon 119 [ß119(GH2)Gly→Ser; HBB: c.358G > A] called Hb Madison-NC.

Expression of the gamma-globin gene is sustained by the cAMP-dependent pathway in beta-thalassaemia.

The present study found that the cyclic adenosine monophosphate (cAMP)-dependent pathway efficiently induced gamma-globin expression in adult erythroblasts, and this pathway plays a role in gamma-globin gene (HBG) expression in beta-thalassaemia. Expression of HBG mRNA increased to about 46% of non-HBA mRNA in adult erythroblasts treated with forskolin, while a cyclic guanosine monophosphate (cGMP) analogue induced HBG mRNA to levels <20% of non-HBA mRNA. In patients with beta-thalassaemia intermedia, cAMP levels were elevated in both red blood cells and nucleated erythroblasts but no consistent elevation was found with cGMP levels. The transcription factor cAMP response element binding protein (CREB) was phosphorylated in nucleated erythroblasts and its phosphorylation levels correlated with HBG mRNA levels of the patients. Other signalling molecules, such as mitogen-activated protein kinases and signal transducers and activators of transcription proteins, were phosphorylated at variable levels and showed no correlations with the HBG mRNA levels. Plasma levels of cytokines, such as erythropoietin, stem cell factor and transforming growth factor-beta were increased in patients, and these cytokines induced both HBG mRNA expression and CREB phosphorylation. These results demonstrate that the cAMP-dependent pathway, the activity of which is augmented by multiple cytokines, plays a role in regulating HBG expression in beta-thalassaemia.