Pathogen reduction with amotosalen/UVA reduces platelet refractoriness in a dog platelet transfusion model.

BACKGROUND AND OBJECTIVES: Pathogen reduction of donor platelets with amotosalen/UVA has been shown to effectively inactivate pathogens and also contaminating white blood cells (WBCs). We wanted to determine whether WBC inactivation could also decrease alloimmune refractoriness to donor platelets. MATERIALS AND METHODS: Platelets were prepared from a donor dog's whole blood, and the platelets were either transfused without modification [standard (STD) platelets] or treated with amotosalen/UVA under conditions modelling the amotosalen/UVA Blood System for human platelets (APR) using either 4 or 3 J/cm2 of UVA exposure. Platelets were transfused weekly from a single donor dog for 8 weeks or until the recipient dog became refractory to their donor's platelets. Antibody samples were drawn weekly and tested against the donor dog's platelets and WBCs (CD8 and B cells). RESULTS: Only 1/7 (14%) dogs that received STD platelets accepted 8 weeks of donor transfusions. Following APR 4 J/cm2 donor transfusions, 3/9 (33%) recipients accepted their donor's transfusions, but only one recipient remained antibody negative. Following APR 3 J/cm2 donor transfusions, the same dose as used for human platelet transfusions, 7/10 (70%) recipients accepted their donor's transfusions, but only two remained antibody negative. CONCLUSION: As a very high percentage of recipient dogs (70%) accepted APR 3 J/cm2 donor transfusions, these data suggest that preventing alloimmune platelet refractoriness may be another benefit of pathogen reduction using amotosalen/UVA.

Effectiveness of Collaborative Care for Depression in Public-Sector Primary Care Clinics Serving Latinos.

OBJECTIVE: Quality improvement interventions for depression care have been shown to be effective for improving quality of care and depression outcomes in settings with primarily insured patients. The aim of this study was to determine the impact of a collaborative care intervention for depression that was tailored for low-income Latino patients seen in public-sector clinics. METHODS: A total of 400 depressed patients from three public-sector primary care clinics were enrolled in a randomized controlled trial of a tailored collaborative care intervention versus enhanced usual care. Social workers without previous mental health experience served as depression care specialists for the intervention patients (N=196). Depending on patient preference, they delivered a cognitive-behavioral therapy (CBT) intervention or facilitated antidepressant medication given by primary care providers or both. In enhanced usual care, patients (N=204) received a pamphlet about depression, a letter for their primary care provider stating that they had a positive depression screen, and a list of local mental health resources. Intent-to-treat analyses examined clinical and process-of-care outcomes at 16 weeks. RESULTS: Compared with patients in the enhanced usual care group, patients in the intervention group had significantly improved depression, quality of life, and satisfaction outcomes (p<.001 for all). Intervention patients also had significantly improved quality-of-care indicators, including the proportion of patients receiving either psychotherapy or antidepressant medication (77% versus 21%, p<.001). CONCLUSIONS: Collaborative care for depression can greatly improve care and outcomes in public-sector clinics. Social workers without prior mental health experience can effectively provide CBT and manage depression care.

A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression.

Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist.

Assessment of nucleic acid modification induced by amotosalen and ultraviolet A light treatment of platelets and plasma using real-time polymerase chain reaction amplification of variable length fragments of mitochondrial DNA.

BACKGROUND: Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS: Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA-treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real-time PCR inhibition assay. RESULTS: PCT induced increasing real-time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION: Our initial findings indicate that an adequately sensitive, quantitative real-time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development.

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

Barriers to depression treatment among low-income, Latino emergency department patients.

Low-income and Latinos use the emergency department (ED) as a primary source of care. Also, the depression prevalence in ED patients is high, making the ED a compelling venue for depression screening and intervention. This study examined barriers and facilitators to depression treatment among low-income, predominantly Latino ED patients. We conducted telephone interviews with 24 ED patients (18-62 years of age, 79 % female) who dropped out of a depression treatment intervention. Using grounded theory, we analyzed perceptions of depression and treatment, and barriers and facilitators to mental health treatment. Although most patients acknowledged signs of depression, there was a lack of readiness to seek help. Patients reported negative perceptions about anti-depressant medication, even if they had no previous use. Barriers to treatment included transportation concerns, employment/unemployment, patient-provider issues, and immigrant documentation. Identified facilitators included consistent provider advice and "talking." This study introduced new misunderstanding and miscommunication barriers.

Defining an EPOR- regulated transcriptome for primary progenitors, including Tnfr-sf13c as a novel mediator of EPO- dependent erythroblast formation.

Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies: Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia). Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.

Peginesatide and erythropoietin stimulate similar erythropoietin receptor-mediated signal transduction and gene induction events.

Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients. To compare signaling effects of peginesatide to recombinant human erythropoietin (rHuEPO), dose-dependent effects on protein phosphorylation and gene expression were evaluated using phosphoproteomics, quantitative signal transduction analyses, and gene profiling. After stimulation with peginesatide or rHuEPO, cell lysates were prepared from UT-7/EPO cells. Liquid chromatography-tandem mass spectrometry and MesoScale arrays were used to quantify phosphorylation events. Transcriptional changes were analyzed using microarrays and quantitative reverse transcription polymerase chain reaction. Peginesatide and rHuEPO were found to regulate the tyrosine phosphorylation of an essentially equivalent set of protein substrates, and modulate the expression of a similar set of target genes. Consistent with their roles in stimulating erythropoiesis, peginesatide and rHuEPO regulate similar cellular pathways.

Dynamic ligand modulation of EPO receptor pools, and dysregulation by polycythemia-associated EPOR alleles.

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects). New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via which mutated EPOR-T polycythemia alleles dysregulate the erythron. Notably, specific new tools also are characterized for studies of EPOR expression, activation, action and metabolism.

Effectiveness of collaborative care in addressing depression treatment preferences among low-income Latinos.

OBJECTIVE: This study assessed treatment preferences among low-income Latino patients in public-sector primary care clinics and examined whether a collaborative care intervention that included patient education and allowed patients to choose between medication, therapy, or both would increase the likelihood that patients received preferred treatment. METHODS: A total of 339 Latino patients with probable depressive disorders were recruited; participants completed a baseline conjoint analysis preference survey and were randomly assigned to receive the intervention or enhanced usual care. At 16 weeks, a patient survey assessed depression treatment received during the study period. Logistic regression models were constructed to estimate treatment preferences, examine patient characteristics associated with treatment preferences, and examine patient characteristics associated with a match between stated preference and actual treatment received. RESULTS: The conjoint analysis preference survey showed that patients preferred counseling or counseling plus medication over antidepressant medication alone and that they preferred treatment in primary care over specialty mental health care, but they showed no significant preference for individual versus group treatment. Patients also indicated that individual education sessions, telephone sessions, transportation assistance, and family involvement were barrier reduction strategies that would enhance their likelihood of accepting treatment. Compared with patients assigned to usual care, those in the intervention group were 21 times as likely to receive preferred treatment. Among all participants, women, unemployed persons, those who spoke English, and those referred by providers were more likely to receive preferred treatment. CONCLUSIONS: Collaborative care interventions that include psychotherapy can increase the likelihood that Latino patients receive preferred care; however, special efforts may be needed to address preferences of working persons, men, and Spanish-speaking patients.

Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia.

OBJECTIVE: To evaluate the preclinical erythropoiesis stimulating properties of Hematide, a novel, PEGylated, synthetic peptide for the treatment of anemia associated with chronic kidney disease and cancer. METHODS: The in vitro activity of Hematide was assessed in competitive binding, proliferation, signal transduction, and apoptosis assays, and in erythroid colony-forming assays with CD34(+) cells purified from human bone marrow. Erythropoiesis and pharmacokinetics were evaluated in rat, monkey, and a rat chronic renal insufficiency (CRI) model following single administration. Erythropoiesis and immunogenicity were also evaluated following repeat administration in rats. RESULTS: Hematide binds and activates the erythropoietin receptor and causes proliferation and differentiation of erythroid progenitor cells. Sustained circulatory persistence of Hematide is observed in rats and monkeys. In a rat CRI model, Hematide exhibited twofold lower clearance than in the normal rat, with hypothesis consistent with Hematide being cleared, at least partially, via the kidney. A dose-dependent rise in hemoglobin (Hgb) and duration of response was observed following single administration in rats and monkeys. Hematide was able to alleviate anemia in an experimental CRI rodent model. Repeat intravenous (IV) and subcutaneous (SC) administration in rats yielded similar erythrogenic responses, with no anti-Hematide antibodies being detected. CONCLUSIONS: Hematide is a potent erythropoiesis stimulating agent with a prolonged half-life and slow clearance times. It is anticipated that similar prolonged clearance and activity will be observed in the clinic, potentially enabling dosing intervals of 3 to 4 weeks that may translate into improved patient convenience for the treatment of anemia.

Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells.

The 1D10 antigen is the target for Hu1D10 (apolizumab), a humanized HLA-DR beta-chain-specific antibody that is currently in clinical trials for hematologic malignancies. We demonstrate that Hu1D10 induces caspase-independent apoptosis following secondary cross-linking in primary chronic lymphocytic leukemia (CLL) cells. Generation of reactive oxygen species (ROS) and signal transduction, as evidenced by phosphorylation of Syk and AKT, were noted. The source of the Hu1D10-induced ROS was examined using the Raji lymphoblastic cell line with engineered defects in the mitochondrial respiratory chain. Hu1D10 treatment of clones with deficient mitochondrial respiration produced ROS suggesting a cytoplasmic source. Administration of ROS scavengers to primary CLL cells prior to Hu1D10 treatment diminished AKT activation. Treatment with Hu1D10 and the phosphatidylinositol 3-kinase inhibitor LY294002 demonstrated in vitro synergy with enhanced apoptosis. In conjunction with an ongoing clinical trial, blood samples were collected following intravenous infusion of Hu1D10 and analyzed for phosphorylation of AKT. Two of 3 patient samples showed a sustained increase in AKT phosphorylation following Hu1D10 administration. These data suggest that Hu1D10 ligation in CLL cells induces death and survival signals for which combination therapies may be designed to greatly enhance efficiency of both Hu1D10 and other class II antibodies in development.

Repeated stress in young and old 5-HT(2C) receptor knockout mice.

Serotonin (5-HT)(2C) receptor null mutant (knockout, KO) mice develop hyperphagia and midlife obesity. Based upon previous observations indicating altered responsiveness to stressful environmental conditions in these mice, we hypothesized that this KO mouse was hyperresponsive to repeated stress. To test this, we examined the effect of two intensities of repeated stress on food intake and body weight in 5-HT(2C) receptor KO and wild-type (WT) mice. The stressors involved daily cage change (including handling) for 3 days then daily restraint for 4 days. On the final day, mice were immediately decapitated after restraint to assess levels of plasma hormones. Two ages were used: young (12 weeks) and old (32-34 weeks). Basally, young KO were prehyperphagic and weighed the same as WT. In the old mice, KO were frankly hyperphagic and heavier than WT. In response to repeated cage change alone, the genotype-specific difference in food intake in the young group was enhanced, whereas in the old group it was diminished. This stressor did not significantly affect body weight change or caloric efficiency with respect to age or genotype. Repeated restraint had little effect on the young mice. However, in the old mice, KO had decreases in relative body weight and caloric efficiency compared with WT. In the old KO mice, adrenocorticotrophic hormone (ACTH), corticosterone and insulin were increased compared with WT mice. Together, these findings indicate that 5-HT(2C) receptor KO mice are hyperresponsive to repeated stress and this effect is influenced by stressor intensity and initial metabolic state of the mouse.

Compulsive behavior in the 5-HT2C receptor knockout mouse.

The efficacy of serotonergic pharmacotherapy indicates that serotonin (5-HT) plays a role in the treatment, if not the etiology, of obsessive-compulsive disorder (OCD). While some clinical evidence implicates 5-HT(2C) receptors in this disorder, a definitive function has yet to be validated. We hypothesized that 5-HT(2C) receptor knockout (KO) mice may display compulsive-like behavior. This paper describes characterization of several distinct, highly organized behaviors in mice lacking functional 5-HT(2C) receptors, which supports a compulsive-like syndrome.Compulsive-like behavior was assessed in male 5-HT(2C) receptor KO and wildtype (WT) mice. Chewing of non-nutritive clay, chewing patterns on plastic-mesh screens, and the frequency of head dipping were measured. 5-HT(2C) receptor KO mice chewed more clay, produced a distinct pattern of "neat" chewing of plastic screens and exhibited reduced habituation of head dipping activity compared to WT mice. We conclude that the 5-HT(2C) receptor null mutant mouse provides a promising model of compulsive behavior and a means to further explore the role of 5-HT in OCD.