Normal Plasma Levels Research Articles

Effect of thrombopoietin‐receptor agonists on a proliferation‐inducing ligand (APRIL) plasma levels in patients with immune thrombocytopaenia

Immune thrombocytopaenia (ITP) is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction and suboptimal platelet production. Patients with ITP have increased plasma levels of a proliferation-inducing ligand (APRIL), a factor that can promote B-cell maturation and survival 1. Two thrombopoietin-receptor agonists [TPO-RA; romiplostim (Nplate®; Amgen, Thousand Oaks, CA, USA) and eltrombopag (Promacta®, Revolade®; GlaxoSmithKline, London, UK)] have recently been approved for the treatment of chronic ITP. They both bind to the thrombopoietin receptor (TPO-R) and activate megakaryopoiesis. We aimed to investigate whether TPO-RA treatment of patients with ITP had any effect on APRIL plasma levels and to compare these results with those observed in ITP patients treated with intravenous immunoglobulins (IVIg). Two groups of chronic ITP patients were included; one group (n = 13) was treated with a TPO-RA (two with romiplostin and 11 with eltrombopag, 46% female; mean age, 63 years) and the other group (n = 17) was treated with IVIg (82% female; mean age, 70 years). The ITP patients were studied before and after responding to therapy, defined as a platelet count ≥30 × 109 l−1 and at least twofold increase over the baseline count. Fiveteen patients with myelodysplastic syndromes who presented thrombocytopaenia (40% female; mean age, 75 years) and 35 healthy control subjects (56% female; mean age, 51 years) were also included for comparison. This study was performed in accordance with the policy of the local Ethics Committee. Blood samples were collected in EDTA. The APRIL concentration was measured by an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA) in platelet-poor plasma. The platelet count of patients with ITP before treatment and of patients with myelodysplastic syndromes were lower than those in the control group (P < 0.001). After responding to the treatments, ITP patients had increased platelet counts (Figure ​(Figure1A1A). Figure 1 (A) Platelet count. (B) Plasma APRIL levels (C) Correlation between a proliferation inducing ligand (APRIL) plasma levels and platelet count. The Wilcoxon matched-pairs signed-ranks test was performed to compare data of patients with immune thrombocytopaenia ... All patients with ITP and thrombocytopaenia showed higher APRIL plasma levels than the control group (P < 0.01, Figure ​Figure1B),1B), which was inversely correlated with platelet count (Figure ​(Figure1C). This1C). This observation supports the proposed pathogenic role of APRIL in the development of this disease 2. Moreover, plasma APRIL levels in ITP patients were also higher than in the myelodysplastic syndrome patients (P < 0.01, Figure ​Figure1B),1B), which suggested that increased APRIL levels were not due to thrombocytopaenia but rather to the mechanism that caused the disease. Plasma levels of APRIL were reduced to control values in patients with ITP who responded to TPO-RA treatment, whereas they remained high after response to IVIg (Figure ​(Figure1B1B). Gu et al. 3 reported normal APRIL plasma levels in patients with ITP with normal platelet counts who had undergone splenectomy or been treated with corticosteroids. In our TPO-RA-treated group, only two of the patients were splenectomized and one was receiving concomitant corticosteroid treatment, so the reduced APRIL levels might be due to another cause. A beneficial effect of TPO-RA treatment on the immune system has been reported 4. Transforming growth factor-β1, an anti-inflammatory cytokine that inhibits B-cell proliferation and antibody production 5, was increased in patients with ITP who responded to TPO-RA treatment. It is therefore tempting to speculate that TPO-RAs possess immunomodulatory activity in addition to their profound effect on megakaryopoiesis. This possibility gives value to this study, despite the small size of the groups included, and gives support to the necessity of performing a study with more patients to elucidate the mechanism involved in the reduction of APRIL levels caused by TPO-RAs.

Hypoadiponectinemia, cardiometabolic comorbidities and left ventricular hypertrophy.

This study was designed to evaluate the prevalence of cardiometabolic comorbidities and the changes in left ventricular geometry and function in 135 subjects subgrouped according to low or normal total adiponectin plasma (ADPN) levels. Left ventricular (LV) internal diameter/height, total LV mass (LVM) and LVM index (LVMI), relative wall thickness (RWT), LV ejection fraction by echocardiography and diastolic parameters by pulsed-wave Doppler were calculated. Body mass index (BMI) (p<0.0001), waist-to-hip ratio (p<0.03), triglycerides (p<0,001), prevalence of obesity (p<0.005), visceral obesity (p<0.003), left ventricular hypertrophy (LVH) (p<0.001), metabolic syndrome (p<0.0003) and coronary artery disease (CAD) (p<0.003) were significantly increased and high-density lipoprotein-cholesterol (p<0.001) was significantly reduced in hypo-ADPN than normal-ADPN subjects. LVM, LVMI, interventricular septum thickness and RWT were significantly (p<0.0001) higher and left ventricular ejection fraction was significantly (p<0.0002) lower in hypo-ADPN than normal-ADPN patients. LVMI correlated directly with BMI (p<0.001), mean blood pressure (p<0.001), metabolic syndrome (MetS) (p<0.001) and inversely with ADPN (p<0.0001). The prevalence of LVH (p<0.001) and CAD (p<0.01) was higher in subjects with normal-ADPN and MetS, while the presence of MetS did not change this finding in hypo ADPN group. Both models of regression analysis indicated that ADPN and BMI resulted independently associated with LVMI. In conclusion, our data seem to indicate that hypoadiponectinemia might be associated with an increased prevalence both of clinical comorbidities and increased LVMI. In this subset of subjects, ADPN and BMI, more than MetS, are able to explain cardiac damage. Accordingly, ADPN might become a new target in the management of cardiometabolic risk.

Hyperhomocysteinaemia, low folate concentrations and MTHFR C677T mutation in abdominal aortic aneurysm.

Homocysteine (Hcy) has been implicated in abdominal aortic aneurysm (AAA). However, the association of Hcy, vitamin B12, and folate in patients with AAA has not been studied in China. This study was conducted with the aim to evaluate the relationship of vitamin B12, folic acid, and Hcy levels in AAA. 463 patients who had AAA were included in this study. 463 control subjects were age- and sex-matched with the patients. In all of the subjects, we evaluated total plasma levels Hcy, vitamin B12, folic acid and the distribution of the C677T methylenetetrahydrofolate reductase (MTHFR) gene mutation. The mean plasma Hcy levels were significantly higher in patients with AAA compared with controls (18.37 ± 6.97 vs. 12.89 ± 4.08 μmol/L, P < 0.001). The frequency of homozygous (TT) genotype in MTHFR C677T mutation was significantly higher in patients with AAA than that in control subjects (19.4 % vs. 11.9 %, P = 0.002). The fasting Hcy correlated negatively with folate (AAA: r = - 0.311, P < 0.01; r = - 0.348, P < 0.01). The aneurysm size was significantly greater (P < 0.001) in patients with hyperhomocysteinemia than that in patients with normal Hcy plasma levels. The size of the AAA had a linear correlation with the plasma Hcy level (r = 0.286; P< 0.001). Serum folate deficiency and hyperhomocysteinemia were associated with an increased risk of AAA in Northeast China. The homozygous (TT) genotype of MTHFR gene mutation may be a crucial hereditary risk factor in AAA.

Spermatid count as a predictor of response to FSH therapy

This study evaluated the predictive power of spermatid count and cytology for assisted reproduction outcome after FSH therapy. A total of 174 men with severe oligozoospermia and normal plasma FSH concentration underwent semen analysis including spermatid count, TUNEL test, FISH analysis for sperm aneuploidies and testicular fine-needle aspiration cytology. Ninety-two men with hypospermatogenesis received FSH therapy for 3months and 82 patients with maturative disturbance or partial obstruction served as controls. Semen was analysed at baseline, after FSH therapy and after 3- and 9-month follow up, and pregnancies were recorded. Subjects not reaching pregnancy at 3-month follow up were recommended assisted reproduction treatment. Spermatid count was related to testicular cytology: spermatid concentrations <0.01, 0.01–0.3 and >0.3×106/ml were predictive of partial obstruction, hypospermatogenesis and maturative disturbance. FSH therapy patients showed increases in sperm number and motility (both P<0.001), allowing some couples to undergo intrauterine insemination instead of IVF. Cumulative pregnancy rate after 12months was higher with FSH therapy (44.6%) than without (22.0%; P=0.002). FSH therapy improved pregnancy rate and sometimes allowed less invasive assisted reproduction treatment in well-selected patients. Spermatid count could represent a new parameter to predict response to FSH therapy.One-hundred seventy-four patients with severe reduction of sperm count and normal sex hormones plasma levels underwent semen analysis with spermatid count, and testicular fine needle aspiration cytologiy (FNAC). Ninety-two men infertile men with reduced sperm production (hypospermatogenesis) were treated with highly purified urofollitropin and 82 patients with sperm maturative defects or partial obstruction of the seminal tract served as controls. After treatment and after the following 3 and 9months all subjects performed a new semen analysis and pregnancies were recorded. Subjects who had not reached spontaneous pregnancy were suggested to undergo assisted reproductive techniques (ARTs). Spermatid count was strongly related to testicular cytology: spermatid concentrations were predictive of partial obstruction, hypospermatogenesis and maturative disturbance respectively. Treated patients showed significant increase in sperm number and motility allowing some couples to undergo easier and less invasive assisted reproductive techniques. The number of pregnancies was significantly higher among treated (44.6%) than untreated couples (22.0%). Our data confirmed that FSH treatment can induce a significant improvemet of pergnancy rate and sometimes allows less invasive ARTs use in well selected severe oligozoospermic patients. Moreover, we suggest that spermatid count can be useful to define tubular status and could represent a new parameter to predict response to FSH therapy.

Quantitation of Insulin Analogue Glargine and Its Two Metabolites M1 and M2 on Triple Quad 6500 and Triple TOF 5600 LC-MS/MS Systems in a Dog Toxicokinetics Study

Glargine is a long lasting bioengineered insulin analogue commonly used in the medical treatment of insulindependent diabetes mellitus. After subcutaneous injection, glargine undergoes enzymatic process generating two metabolites, M1 and M2. Quantitative evidences of their presence and concentration after doping multiple concentrations are important for clinical study. Such information can also help to understand the pharmacokinetics, pharmacodynamics, and toxicology during the insulin drug development. We developed and validated a high throughput analytical method coupled with solid-phase extraction (SPE) and LC-MS/MS for the quantitation of intact insulin in plasma samples for use in pre-clinical and clinical studies. The multiple-reaction-monitoring (MRM) experiments performed on a triple-quadruple mass spectrometry instrument were used for quantitation. A 3.5 min UHPLC method was developed to achieve high throughput. An eight-point standard calibration curve with concentration from 0.2 ng/mL to 20 ng/mL was constructed corresponding to the normal non-fasting insulin levels in plasma. The acceptance criteria for values of accuracy, precision, and linearity of the quantitation curve were developed in accordance to the bioanalytical method validation guidance published by Food and Drug Administration (FDA) and European Medicines Agency (EMA). This validated MRM method was applied for toxicity study of insulin analogues in dog. The toxicokinetic results were reported. Moreover, we compared the MRM results to the MS quantitation results acquired on a hybrid quadruple-quadruple time-of-flight (QqTOF) and showed the high resolution instrument can be an option for peptide and biotherapeutic protein quantitation.

Vitamin E is essential for Purkinje neuron integrity

α-Tocopherol (vitamin E) is an essential dietary antioxidant with important neuroprotective functions. α-Tocopherol deficiency manifests primarily in neurological pathologies, notably cerebellar dysfunctions such as spinocerebellar ataxia. To study the roles of α-tocopherol in the cerebellum, we used the α-tocopherol transfer protein for the murine version (Ttpa−/−) mice which lack the α-tocopherol transfer protein (TTP) and are a faithful model of vitamin E deficiency and oxidative stress. When fed vitamin E-deficient diet, Ttpa−/− mice had un-detectable levels of α-tocopherol in plasma and several brain regions. Dietary supplementation with α-tocopherol normalized plasma levels of the vitamin, but only modestly increased its levels in the cerebellum and prefrontal cortex, indicating a critical function of brain TTP. Vitamin E deficiency caused an increase in cerebellar oxidative stress evidenced by increased protein nitrosylation, which was prevented by dietary supplementation with the vitamin. Concomitantly, vitamin E deficiency precipitated cellular atrophy and diminished dendritic branching of Purkinje neurons, the predominant output regulator of the cerebellar cortex. The anatomic decline induced by vitamin E deficiency was paralleled by behavioral deficits in motor coordination and cognitive functions that were normalized upon vitamin E supplementation. These observations underscore the essential role of vitamin E and TTP in maintaining CNS function, and support the notion that α-tocopherol supplementation may comprise an effective intervention in oxidative stress-related neurological disorders.

Heparin-binding protein (HBP) in critically ill patients with influenza A (H1N1) infection

AbstractHeparin-binding protein (HBP) is an inducer of vascular endothelial leakage in severe infections. Fluid accumulation into alveoli is a general finding in acute respiratory distress syndrome (ARDS). Severe acute respiratory failure with ARDS is a complication of influenza A(H1N1) infection. Accordingly, we studied the HBP levels in critically ill patients with infection of influenza A(H1N1).Critically ill patients in four intensive care units (ICUs) with polymerase chain reaction (PCR) confirmed infection of influenza A(H1N1) were prospectively evaluated. We collected clinical data and blood samples at ICU admission and on day 2. Twenty-nine patients participated in the study. Compared with normal plasma levels, the HBP concentrations were highly elevated at baseline and at day 2: 98 ng/mL (62–183 ng/mL) and 93 ng/mL (62–271 ng/mL) (p 0.876), respectively. HBP concentrations were correlated with the lowest ratio of partial pressure of oxygen in arterial blood to fraction of inspired oxygen (PF ratio) during the ICU stay (rho = −0.321, p <0.05). In patients with and without invasive mechanical ventilation, the baseline HBP levels were 152 ng/mL (72–237 ng/mL) and 83 ng/mL (58–108 ng/mL) (p 0.088), respectively. The respective values at day 2 were 223 ng/mL (89–415 ng/mL) and 81 ng/mL (55–97 ng/mL) (p <0.05). The patients with septic shock/severe sepsis (compared with those without) did not have statistically significant differences in HBP concentrations at baseline or day 2. HBP concentrations are markedly elevated in all critically ill patients with influenza A(H1N1) infection. The increase in HBP concentrations seems to be associated with more pronounced respiratory dysfunction.

Immune-Mediated Activation Of Coagulation In Patients With Stevens Johnson Syndrome/Toxic Epidermal Necrolysis

BackgroundStevens Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) are specific drug hypersensitivity reactions initiated by cytotoxic T-lymphocytes. Augmentation of T-cell activation by positive co-stimulation and Major Histocompatibility Complex-restricted presentation of the culprit drug triggers T-cell activation following interaction with T-cell receptors. This T-cell activation results in expression of cytokines including TNF-α, interferon-γ, granzyme B and granulysin from NK cells. Besides their role in causing SJS/TEN, these cytokines cause coagulation activation. Materials & MethodsFollowing an Institutional Review Board approved protocol blood samples were obtained from subjects suspected of SJS/TEN and normal healthy volunteers (n=2). Confirmatory biopsies were performed in all SJS/TEN subjects which confirmed SJS/TEN in 4 subjects and were negative in 7 subjects. Cytokine levels were measured using the cerebral II array biochip from Randox Laboratories Limited (Crumlin, UK). Besides cerebral array II, other parameters such as thrombin-antithrombin (TAT) complexes (Dade®, Marburg, Germany), fibrinopeptide A (F1.2, Dade®), plasminogen activator inhibitor-1 (PAI-1, Diagnostica Stago® and STACHROME antithrombin (Stago®), ZYMUPHEN platelet microparticles activity (Hyphen® Biomed (Neuville-sur-Oise, France), and HEMOCLOT protein C (Stago®) were measured using ELISA kits as per manufacturers' instructions. ResultsCompared to the Normal Human Plasma, the IL-4, IL-6, TNF-α, and MCP-1 levels were increased significantly in biopsy-confirmed SJS/TEN patients (see table). Other parameters measured including IL-2, IL-8, IL-10, VEGF, INF-γ, IL-1α, IL1-β and EGF were not significantly increased. A marked increase in the TAT complexes (6.3±5.9 µg/ml), F1.2 (430.4±202.4 pmol/L), platelet microparticles (13.1±9.3 nM) and protein C levels (90.5±63.4%) with a corresponding decrease in PAI-1 (53.3±18.8ng/ml) and antithrombin levels (80.7±42.4%) compared to normal human plasma were also observed. Biopsy-negative SJS/TEN subjects with less pronounced inflammatory stimulus, demonstrated mild elevation in cytokine levels of IL-4 (1.95±0.59 pg/ml), IL-6 (29.81±25.18 pg/ml), TNF-α (7.20±5.04 pg/ml) and MCP-1 (265.10±159.09 pg/ml)).TableInflammatory biomarkers in SJS/TEN patients compared to control plasmaBiomarkerIL-4 (pg/ml)IL-6 (pg/ml)TNF-α (pg/ml)MCP-1 (pg/ml)SJS/TEN-Mean1.6950.53.20222.74Control Plasma1.420.450.7725.28p-value0.00940.02760.01380.0025 ConclusionsT-cell activation and release of cytokines especially TNF-α and granzyme B causes immune-mediated activation of coagulation with increase in TAT, MCP-1, F1.2 and platelet microparticles and corresponding decrease of protein C, antithrombin, and PAI-1. These alterations in coagulation may progress to sepsis associated coagulopathy and overt disseminated intravascular coagulation. Disclosures:No relevant conflicts of interest to declare.

Rhabdomyolysis in an elderly multitreated patient: Multiple drug interactions after statin withdrawal

Rhabdomyolysis precipitated by multitherapy is most frequently described during statin treatment, due to impairment of statin clearance by drugs sharing cytochrome P450 biotransformation pathway. Modulation of membrane transporters for drug efflux, operated by substrates, can also affect drugs' tissue levels. We report rhabdomyolysis in an elderly patient, in multitreatment with different potentially myotoxic medications, taking place seven months after atorvastatin discontinuation. Affected by ischaemic heart disease, arterial hypertension and dementia-related behaviour disturbances, the patient was receiving angiotensin 2-receptor inhibitors, beta-blockers, vasodilators, diuretics, salycilates, allopurinol, proton pump inhibitors, antipsychotics and antidepressants. He had taken atorvastatin for 14years, with constantly normal creatine-kinase plasma levels. Two months after addition of the antianginal drug ranolazine, creatine-kinase mildly increased and atorvastatin was withdrawn. Nonetheless, creatine-kinase progressively rose, with severe weakness and rhabdomyolysis developing seven months later. Muscle biopsy showed a necrotizing myopathy with no inflammation or autoimmune changes. After ranolazine withdrawal, creatine-kinase and myoglobin returned to normal levels and strength was restored. Several psychotropic and cardiovascular medications prescribed to the patient share either cytochrome P450 biotransformation and permeability-glycoprotein efflux transport. In the event of cardiovascular/neuropsychiatric polypharmacy in geriatric patients, the risk of muscle severe adverse effects from pharmacokinetic drug–drug interaction should be considered beyond the direct myotoxicity of statins.

Anti-ghrelin immunoglobulins modulate ghrelin stability and its orexigenic effect in obese mice and humans

Obese individuals often have increased appetite despite normal plasma levels of the main orexigenic hormone ghrelin. Here we show that ghrelin degradation in the plasma is inhibited by ghrelin-reactive IgG immunoglobulins, which display increased binding affinity to ghrelin in obese patients and mice. Co-administration of ghrelin together with IgG from obese individuals, but not with IgG from anorectic or control patients, increases food intake in rats. Similarly, chronic injections of ghrelin together with IgG from ob/ob mice increase food intake, meal frequency and total lean body mass of mice. These data reveal that in both obese humans and mice, IgG with increased affinity for ghrelin enhances ghrelin’s orexigenic effect, which may contribute to increased appetite and overeating.

Repression of GH signaling: One extended life to live!

Repression of GH signaling: One extended life to live!

Secretion of adiponectin from mouse aorta and its role in cold storage-induced vascular dysfunction

Availability of adiponectin plays a crucial role in cardiovascular function. The present study was conducted to evaluate the presence, alterations and impact of the various adiponectin isoforms in vascular tissue under clinically relevant in vitro conditions (cold storage). Presence of various adiponectin isoforms in vascular smooth muscle cells and their regulation during cold storage was evaluated by PCR, western blot, ELISA and immunohistochemistry. The impact of the various isoforms for vessel preservation was assessed using isometric force measurement as an in vitro assay for vascular function. Adiponectin is expressed in smooth muscle cells from murine aortae and human saphenous veins. Following 2 days of cold storage adiponectin mRNA expression in mouse aorta is reduced, which appears to be regulated indirectly by miR-292-3p. Despite the reduced mRNA expression, adiponectin accumulated in cold storage supernatant over 2 days indicating a net release of adiponectin. Two days of cold storage resulted in an impairment of endothelium-dependent relaxation which was prevented by addition of full-length adiponectin in concentrations similar to normal plasma levels during storage. In contrast, addition of recombinant adiponectin which is unable to form high order multimers failed to improve vessel function. High concentrations (20 μg/mL) of this trimeric isoform even reduced the vasorelaxation response and facilitated uncoupling of endothelial nitric oxide synthase. Endothelial injury by cold storage may partly be prevented by addition of high-molecular-weight adiponectin. This effect may support graft patency to avoid coagulation- and atherosclerosis-associated impairment of perfusion.

Increased Occurrence of Valproic Acid-Induced Hyperammonemia in Carriers of T1405N Polymorphism in Carbamoyl Phosphate Synthetase 1 Gene

Numerous cases of severe and life-threatening hyperammonemia (HA) related to the treatment of epileptic seizures with valproic acid (VPA) have been previously reported in the medical literature. The aim of this prospective, multicenter study was to verify the putative association between T1405 polymorphism and occurrence of VPA-induced HA in the cohort of 142 adult Caucasian patients with epilepsy treated with VPA for at least 1 year and with normal liver functions. The nonsynonymous T1405N polymorphism genotyping was performed by real-time TaqMan PCR genotyping. In addition to plasma ammonia level, concentrations of liver enzymes and total VPA were measured in plasma with standard laboratory methods. HA (defined as ammonia plasma level >65 μmol/L) was observed in total of 11 (7.7%) of patients treated with VPA, and the carrier status for the investigated polymorphism was significantly (P = 0.009, odds ratio 5.4 with 95% confidence interval of 1.58–18.43) associated with the occurrence of HA. The results of this study support a notion that in the Caucasian patients with epilepsy undergoing VPA therapy, a T1405N (4217C > A, rs1047891) nonsynonymous variant was a significant risk factor for the occurrence of HA, even in patients with normal plasma levels of VPA.

Decreased tissue factor pathway inhibitor (TFPI)‐dependent anticoagulant capacity in patients with cirrhosis who have decreased protein S but normal TFPI plasma levels

Protein S acts as a cofactor for tissue factor pathway inhibitor (TFPI) in the down regulation of thrombin generation, and acquired and congenital protein S deficiencies are associated with a concomitant TFPI deficiency. In contrast, in patients with liver diseases, decreased protein S, but normal or increased levels of TFPI have been reported. We compared TFPI and protein S plasma levels between 26 patients with cirrhosis and 20 healthy controls and found that TFPI levels were comparable between patients (111 ± 38%) and controls (108 ± 27%), despite reduced protein S levels (74 ± 23% in patients vs. 98 ± 10% in controls). Subsequently, we quantified the activity of the TFPI-protein S system by measuring thrombin generation in the absence and presence of neutralizing antibodies to protein S or TFPI. Ratios of peak thrombin generation in the absence and presence of these antibodies were calculated. Both the protein S and the TFPI ratios were increased in patients with cirrhosis compared to controls. Protein S ratios were (0·62 [0·08-0·93] in patients vs. 0·32 [0·20-0·54] in controls; TFPI ratios were 0·50 [0·05-0·90] in patients vs. 0·18 [0·11-0·49] in controls). Thus, although the acquired protein S deficiency in patients with cirrhosis is not associated with decreased TFPI levels, the TFPI/protein S anticoagulant system is functionally impaired.

High-density lipoprotein cholesterol affects early endothelial progenitor cell number and endothelial function in obese women

In order to improve our understanding of high-density lipoprotein cholesterol (HDL-C) cardiovascular (CV) impact in obesity, the association of HDL-C plasma level with circulating early endothelial progenitor cell (early-EPC) number and endothelium-dependent vasodilatation (EDV) in obese women with normal or high low-density lipoprotein cholesterol (LDL-C) plasma levels was evaluated. One hundred thirteen obese female subjects and a control group of 78 healthy female subjects were recruited. Circulating early-EPC were assessed by single- and two-color flow cytometric analyses with a fluorescence activated cell sorting (FACScan) flow cytometer. EDV was evaluated as response to ischemia by strain gauge plethysmography. Both early-EPC number and EDV were significantly decreased in obese women compared with the control group. Obese women with low HDL-C showed a further decrease of early-EPC and EDV in the presence of both high or normal LDL-C plasmatic levels. In the normal HDL-C level subgroup, hypercholesterolemic and nonhypercholesterolemic subjects showed no difference in early-EPC number, whereas slight EDV impairment was present in hypercholesterolemic subjects. In obese women, low HDL-C is associated to decreased early-EPC number and impaired EDV, suggesting the need to assess whether evaluation of early-EPC and EDV may increase HDL-C prognostic value in the stratification of CV risk.

Adult sea lamprey tolerates biliary atresia by altering bile salt composition and renal excretion.

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia, as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the postmetamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic, with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (∼10 μM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ∼35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72-hour period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts, and BSP, were predominantly excreted by way of the kidney, with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney, with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis. (HEPATOLOGY 2013;57:2418-2426).

The Roles of Fibroblast Growth Factor (FGF)-23, α-Klotho and Furin Protease in Calcium and Phosphate Homeostasis : A Mini-Review.

The roles of calcitonin, parathormone and calcitriol in the regulation of plasma calcium and phosphate are well-established. However, in autosomal-dominant hypophosphatemic rickety patients, studies have revealed normal plasma levels of calcium, associated with normal thyroid and parathyroid functions, but decreased levels of phosphate and calcitriol despite adequate reserves of vitamin D. Also, in tumoral calcinosis, persistent hyperphosphatemia with increased levels of 1,25(OH)2D3 have been observed. These studies indicate the involvement of factors other than the ones already known. The first decade of this century/millennium has led to the discovery of the involvement of fibroblast growth factor-23, furin protease and α-klotho in the homeostasis of calcium and phosphate, which is the subject of this mini-review.

Genetic variants associated with protein C levels

Normal protein C (PC) plasma levels range widely in the general population. Factors influencing normal PC levels are thought to influence the risk of venous thrombosis. Little is known about the underlying genetic variants. We performed a genome scan of normal PC levels to identify genes that regulate normal PC levels. We performed a variance components linkage analysis for normal PC levels in 275 individuals from a single, large family. We then sequenced candidate genes under the identified linkage peak in eight family members: four with high and four with low, but normal, PC levels. For variants showing a difference in carriers between those with high and low PC levels, we re-evaluated linkage in the 275 family members conditional on the measured genotype effect. Genotype-specific mean PC levels were determined using likelihood analysis. Findings were replicated in the Leiden Thrombophilia Study (LETS). We identified a quantitative trait locus at chromosome 5q14.1 affecting normal PC plasma level variability. Next-generation sequencing of 113 candidate genes under the linkage peak revealed four SNPs in BHMT2, ACOT12, SSBP2 and XRCC4, which significantly increased PC levels in our thrombophilic family, but not in LETS. We identified four genes at chromosome 5q14.1 that might influence normal PC levels. BHMT2 seems the most likely candidate to regulate PC levels via homocysteine, a competitive inhibitor to thrombin. Failure to replicate our findings in LETS might be due to differences between the studies in genetic background and linkage disequilibrium patterns.

Alpha-1 Antitrypsin Augmentation Therapy

The therapy of alpha-1 antitrypsin deficiency (AATD) is an example of a medical triumph over a common hereditary disease. Based on the understanding of the pathogens of the disease as a deficiency in liver production of alpha-1 antitrypsin (AAT) resulting from inherited genetic variation in both parental AAT genes, the knowledge that A1AT functions primarily to inhibit neutrophil elastase (NE), and the observation that NE instilled into the lung of experimental animals resulted in emphysema, the concept evolved that the pulmonary manifestations of the disease could be halted by intermittent intravenous infusions of AAT purified from pooled human plasma. Following preliminary clinical studies in the academic community, and then pharmaceutical company development of large scale purification of human AAT, the FDA approved the use of weekly AAT augmentation therapy for AATD following a clinical trial which demonstrated that weekly infusions would raise to normal plasma and lung epithelial fluid levels of AAT in AAT-deficient individuals. The therapy is now used worldwide to treat AATD, the only pulmonary genetic disease with effective therapy for all affected individuals.

The effect of pre-storage whole-blood leukocyte reduction on cytokines/chemokines levels in autologous CPDA-1 whole blood

BackgroundIn this study, we aimed to investigate the effectiveness of pre-storage leukocyte filtration of autologous blood (AB), especially focusing on the cytokines/chemokines accumulation on blood products. Materials and methodsAfter approval of the ethics committee of the University of Tokyo, a total of 26 orthopedic patients, who donated AB prior to surgery after informed consent, were enrolled. The effects of filtration on blood cell counts were analyzed, and the accumulation of cytokines and chemokines were measured on pre- and post-leukoreduced (LR) samples, using the Luminex system. The time-dependent changes of the cytokines/chemokines and the effect of the filtration on their concentration were analyzed, and compared with the normal plasma levels reported in the literature. ResultsLR effectively reduced the number of leukocytes and platelets, without affecting that of red cells. The concentration of most of the cytokines/chemokines analyzed, except the EGF, sCD40-L and sFas-L, decreased time-dependently of storage or did not change in pre-LR samples. However, EGF, sCD40L and sFas-L were significantly reduced by LR. Some, such as IL-8 and RANTES, were also importantly decreased by LR, and others, such as IL-1β and TNF-α, were not significantly affected by LR. ConclusionsLeukocyte filtration effectively removes platelets and leukocytes from AB, thus preventing the accumulation of cytokines/chemokines. Since adverse effects due to AB transfusion, although rare, are observed, there is need to consider the implementation of pre-storage leukocyte reduction (PSLR) for AB.