Systemic and regional hemorheological consequences of warm and cold hind limb ischemia-reperfusion in a canine model

We have studied systemic and regional changes in hemorheological parameters after complete acute limb ischemia and reperfusion (I/R) in 24 mongrel dogs. Unilateral cooled and non-cooled vascular ischemia (3 h)-reperfusion (4 h), and sham-operations were performed. Blood samples were collected from the excluded region, during reperfusion and for 5 days. Whole blood and plasma viscosity (WBV, PV), relative cell transit time (RCTT) of erythrocytes, fibrinogen level and hematological parameters were determined. In I/R groups WBV of excluded blood was significantly higher compared to the base (p < 0.05), and RCTT increased during the reperfusion. On 2nd-3rd days RCTT increased significantly in both I/R groups. In each group PV and fibrinogen showed continuous increase during the postoperative period, prominently in cooled I/R group, and furthermore WBV corrected for hematocrit (40%) was the highest in cooled I/R group. These suggest that surgical acute limb I/R may cause hemorheological changes, which are more serious after cooling. (Grants: OTKA-T032571, 6003/1/2001/ETT.)     

Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs

Hepcidin is the principal iron regulatory hormone and its overproduction contributes to anemia of inflammation (AI). In vitro, hepcidin binds to and induces the degradation of the exclusive iron exporter ferroportin. We explored the effects and distribution of synthetic hepcidin in the mouse. A single intraperitoneal injection of hepcidin caused a rapid fall of serum iron in a dose-dependent manner, with a 50-microg dose resulting in iron levels 80% lower than in control mice. The full effect was seen within only 1 hour, consistent with a blockade of iron export from tissue stores and from macrophages involved in iron recycling. Serum iron remained suppressed for more than 48 hours after injection. Using radiolabeled hepcidin, we demonstrated that the serum concentration of hepcidin at the 50-microg dose was 1.4 microM, consistent with the inhibitory concentration of 50% (IC50) of hepcidin measured in vitro. Radiolabeled hepcidin accumulated in the ferroportin-rich organs, liver, spleen, and proximal duodenum. Our study highlights the central role of the hepcidin-ferroportin interaction in iron homeostasis. The rapid and sustained action of a single dose of hepcidin makes it an appealing agent for the prevention of iron accumulation in hereditary hemochromatosis.     

Iron homeostasis in pregnancy and spontaneous abortion

During pregnancy, iron requirements are increased to support maternal erythropoietic expansion and fetal growth and development. To meet these requirements, dietary iron absorption increases, and available iron stores are mobilized. These adjustments are thought to be in large part mediated by the iron-regulatory hormone hepcidin, which controls the concentrations of ferroportin, the sole exporter of iron into the extracellular fluid and blood plasma. Hepcidin regulation of iron availability during healthy and abnormal pregnancies is not well understood. In our cross-sectional study, we compared hepcidin, iron and hematological parameters between nonpregnant control women, healthy pregnant women in the first and second trimester, and women with spontaneous abortion in the first trimester. We found that in healthy pregnancy, hepcidin increased in the first trimester compared with nonpregnant women, but then decreased during the second trimester. The second trimester hepcidin levels decreased despite stable serum iron concentrations, suggesting active suppression of hepcidin, presumably to enhance iron availability as iron demand increases. In women with spontaneous abortion during the first trimester, hepcidin, serum iron, and ferritin concentrations were all increased compared with the first trimester healthy pregnancy. Although the specific mechanisms remain to be determined, our findings demonstrate that maternal hepcidin is regulated by signals related to the progression of pregnancy, and that pregnancy loss is associated with profound changes in maternal iron metabolism. These observations highlight the existence of fetoplacental signals that modulate maternal iron homeostasis.     

Measurement of fecal glucocorticoids in parrotfishes to assess stress

Coral reefs are in decline worldwide from a combination of natural and human forces. The environmental compromises faced by coral reef habitats and their associated fishes are potentially stressful, and in this study we examined the potential for assessing stress levels in coral reef fish. We determined the feasibility of using fecal casts from parrotfishes for remote assessment of stress-related hormones (cortisol and corticosterone), and the response of these hormones to the stress of restraint and hypoxia. Measurement of these hormones in fecal extracts by high performance liquid chromatography (HPLC) was validated using mass spectrometry, chemical derivitization, and radioactive tracer methods. In aquarium-adapted parrotfish, baseline levels of cortisol and corticosterone averaged 3.4+/-1.1 and 14.8+/-2.8 ng/g feces, respectively, across 32 days. During 13 days of periodic stress these hormones, respectively, average 10.8-fold and 3.2-fold greater than baseline, with a return to near baseline during a 23-day follow-up. Testosterone was also measured as a reference hormone which is not part of the stress-response axis. Levels of this hormone were similar across the study. These fecal hormones were also measured in a field study of parrotfish in 10 fringing coral reef areas around the Caribbean Island of St. John, US Virgin Islands. Extracts of remotely collected fecal casts of three parrotfish species revealed no difference in respective average hormone levels among these species. Also, there was no difference in respective hormone levels between aquarium and field environments. However, levels of both cortisol and corticosterone, but not testosterone, were elevated in two of the 10 reef sites surveyed. This study demonstrates that parrotfish fecals can be collected in aquarium and field conditions and that steroid hormones in these fecals can be extracted and reliably measured. The study also demonstrates that cortisol and corticosterone in parrotfish fecals can be used as an indicator of the stress-response which is unlikely to be masked by intrinsic variability in the sample source, environment or methodology.     

The effects of renal ischemia-reperfusion on hemorheological factors: preventive role of allopurinol

Changes in hemorheological parameters were studied in dogs following unilateral renal artery clamping (45-minute ischemia then reperfusion), with and without preoperative administration of allopurinol. Sham-operated animals were also evaluated. Blood samples were collected preoperatively, at beginning and at 30, 60 and 120 minutes of reperfusion, then on the 1st, 3rd, 5th and 7th days. Filtration properties of erythrocytes (relative cell transit time, RCTT), whole blood and plasma viscosity (WBV, PV), fibrinogen level and hematology parameter were determined. RCTT significantly increased for both ischemic groups at 30 minutes of reperfusion, and remained elevated on the 1st and 2nd postoperative days; these changes were abolished by allopurinol pretreatment. WBV and hematocrit increased on the 1st day, and PV and fibrinogen level showed elevation on 1st-5th postoperative days. We thus conclude that decreases of RBC deformability (i.e., higher RCTT) were characteristic and specific on early postoperative days after renal ischemia-reperfusion and that these alterations were prevented by pre-ischemia administration of allopurinol.     

Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway, which utilizes beta-catenin to relay the signal, have been demonstrated to regulate HSC function. In this study, we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation, such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data, we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway, resulting in a pool of quiescent HSCs.     

Alcohol screening and brief counseling in a primary care hypertensive population: a quality improvement intervention

Aims To determine the effect of an intervention to improve alcohol screening and brief counseling for hypertensive patients in primary care. Design Two‐year randomized, controlled trial. Setting/participants Twenty‐one primary care practices across the United States with a common electronic medical record. Intervention To promote alcohol screening and brief counseling. Intervention practices received site visits from study personnel and were invited to annual network meetings to review the progress of the project and share improvement strategies. Measurements Main outcome measures included rates of documented alcohol screening in hypertensive patients and brief counseling administered in those diagnosed with high‐risk drinking, alcohol abuse or alcohol dependence. Secondary outcomes included change in blood pressure among patients with these diagnoses. Findings Hypertensive patients in intervention practices were significantly more likely to have been screened after 2 years than hypertensive patients in control practices [64.5% versus 23.5%; adjusted odds ratio (OR) = 8.1; 95% confidence interval (CI) 1.7–38.2; P < 0.0087]. Patients in intervention practices diagnosed with high‐risk drinking, alcohol abuse or alcohol dependence were more likely than those in control practices to have had alcohol counseling documented (50.5% versus 29.6%; adjusted OR = 5.5, 95% CI 1.3–23.3). Systolic (adjusted mean decline = 4.2 mmHg, P = 0.036) and diastolic (adjusted mean decline = 3.3 mmHg, P = 0.006) blood pressure decreased significantly among hypertensive patients receiving alcohol counseling. Conclusions Primary care practices receiving an alcohol‐focused intervention over 2 years improved rates of alcohol screening for their hypertensive population. Implementation of alcohol counseling for high‐risk drinking, alcohol abuse or alcohol dependence also improved and led to changes in patient blood pressures.     

Blockade of interleukin-6 signalling with siltuximab enhances melphalan cytotoxicity in preclinical models of multiple myeloma

Signalling through the interleukin (IL)-6 pathway induces proliferation and drug resistance of multiple myeloma cells. We therefore sought to determine whether the IL-6-neutralizing monoclonal antibody siltuximab, formerly CNTO 328, could enhance the activity of melphalan, and to examine some of the mechanisms underlying this interaction. Siltuximab increased the cytotoxicity of melphalan in KAS-6/1, INA-6, ANBL-6, and RPMI 8226 human myeloma cell lines (HMCLs) in an additive-to-synergistic manner, and sensitized resistant RPMI 8226.LR5 cells to melphalan. These anti-proliferative effects were accompanied by enhanced activation of drug-specific apoptosis in HMCLs grown in suspension, and in HMCLs co-cultured with a human-derived stromal cell line. Siltuximab with melphalan enhanced activation of caspase-8, caspase-9, and the downstream effector caspase-3 compared with either of the single agents. This increased induction of cell death occurred in association with enhanced Bak activation. Neutralization of IL-6 also suppressed signalling through the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased phosphorylation of Akt, p70 S6 kinase and 4E-BP1. Importantly, the siltuximab/melphalan regimen demonstrated enhanced anti-proliferative effects against primary plasma cells derived from patients with myeloma, monoclonal gammopathy of undetermined significance, and amyloidosis. These studies provide a rationale for translation of siltuximab into the clinic in combination with melphalan-based therapies.     

The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow-derived endothelial cells in brains of mice following cerebral ischemia

Granulocyte colony-stimulating factor (G-CSF) induces proliferation of bone marrow–derived cells. G-CSF is neuroprotective after experimental brain injury, but the mechanisms involved remain unclear. Stem cell factor (SCF) is a cytokine important for the survival and differentiation of hematopoietic stem cells. Its receptor (c-kit or CD117) is present in some endothelial cells. We aimed to determine whether the combination of G-CSF/SCF induces angiogenesis in the central nervous system by promoting entry of endothelial precursors into the injured brain and causing them to proliferate there. We induced permanent middle cerebral artery occlusion in female mice that previously underwent sex-mismatched bone marrow transplantation from enhanced green fluorescent protein (EGFP)–expressing mice. G-CSF/SCF treatment reduced infarct volumes by more than 50% and resulted in a 1.5-fold increase in vessel formation in mice with stroke, a large percentage of which contain endothelial cells of bone marrow origin. Most cells entering the brain maintained their bone marrow identity and did not transdifferentiate into neural cells. G-CSF/SCF treatment also led to a 2-fold increase in the number of newborn cells in the ischemic hemisphere. These findings suggest that G-CSF/SCF treatment might help recovery through induction of bone marrow–derived angiogenesis, thus improving neuronal survival and functional outcome.     

Erythropoiesis: Review Article: Slow and Steady Wins The Race? Progress in the Development of Vectors for Gene Therapy of beta-Thalassemia and Sickle Cell Disease

The cloning of the human beta-globin genes more than 20 years ago led to predictions that beta-thalassemia and sickle cell disease would be among the first monogenic diseases to be successfully treated by gene replacement therapy. However, despite the world-wide enrollment of more than 3,000 patients in approved gene transfer protocols, none have involved therapy for these diseases. This has been due to several technical hurdles that need to be overcome before gene replacement therapy for beta-thalassemia and sickle cell disease can become practical. These problems include inefficient transduction of hematopoietic stem cells and an inability to achieve consistent, long-term, high-level expression of transferred beta-like globin genes with current gene transfer vectors. In this review we highlight the relationships between understanding the fundamental mechanisms of beta-globin gene locus function and basic vector biology and the development of strategies for beta-globin gene replacement therapy. Despite slow initial progress in this field, recent advances in a variety of critical areas provide hope that clinical trials may not be far away.