Retrospectively gated MRI for in vivo assessment of endothelium‐dependent vasodilatation and endothelial permeability in murine models of endothelial dysfunction

Endothelial dysfunction is linked to impaired endothelial‐dependent vasodilatation and permeability changes. Here, we quantify both of these phenomena associated with endothelial dysfunction by MRI in vivo in mice. Endothelial function was evaluated in the brachiocephalic artery (BCA) and left carotid artery (LCA) in ApoE/LDLR^?/? and high‐fat diet (HFD)‐fed mice as compared with control mice (C57BL/6J). The 3D IntraGate® FLASH sequence was used for evaluation of changes in vessels’ cross‐sectional area (CSA) and volume following acetylcholine (Ach) administration. Evaluation of endothelial permeability after administration of contrast agent (Galbumin, BioPAL) was based on the variable flip angle method for the assessment of parameters based on the relaxation time (T₁) value. In order to confirm the involvement of nitric oxide (NO) in response to Ach, L‐NAME‐treated mice were also analyzed. To confirm that endothelial permeability changes accompany the impairment of Ach‐dependent vasodilatation, permeability changes were analyzed in isolated, perfused carotid artery. In C57BL/6J mice, Ach‐induced vasodilatation led to an approximately 25% increase in CSA in both vessels, which was temporarily dissociated from the effect of Ach on heart rate. In ApoE/LDLR^?/? or HFD‐fed mice Ach induced a paradoxical vasoconstriction that amounted to approximately 30% and 50% decreases in CSA of BCA and LCA respectively. In ApoE/LDLR^?/? and HFD‐fed mice endothelial permeability in BCA was also increased (fall in T₁ by about 25%). In L‐NAME‐treated mice Ach‐induced vasodilatation in BCA was lost. In isolated, perfused artery from ApoE/LDLR^?/? mice endothelial permeability was increased. MRI‐based assessment of endothelium‐dependent vasodilatation induced by Ach and endothelial permeability using a retrospectively self‐gated 3D gradient‐echo sequence (IntraGate® FLASH) enables the reliable detection of systemic endothelial dysfunction in mice and provides an important tool for the experimental pharmacology of the endothelium in murine models of diseases in vivo. Copyright © 2016 John Wiley & Sons, Ltd.     

Novel mutations and polymorphisms in genes causing hereditary hemorrhagic telangiectasia

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder caused by mutations in Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes. We performed molecular characterization in clinically affected probands of 31 HHT families and detected a total of 28 different mutations in the two genes, including four shared by more than one family. Twelve mutations were identified in the ENG gene, six of which were novel and comprised two nonsense mutations in exons 6 and 8, deletions in exons 5 and 11, and splice site mutations in exon 12 and intron 8. Eleven of sixteen mutations identified in the ALK1 gene were novel single base pair substitutions in exons 4, 7, 8, and 9. We also describe the first de novo ALK1 mutation that causes a previously unreported c.1133C>A substitution of a highly conserved residue (p.P378H). The proband and his two daughters, who also carried the familial mutation, all suffered from gastrointestinal (GI) bleeding. In addition, we report seven newly identified polymorphisms and summarize all known ones in both genes.     

A two year observation of the process of applying recombinant IGF-1 to treat short stature in children with primary IGF-1 deficiency -- case reports of 3 patients

Growth deficiency is one of the most frequent causes of referral to Endocrinology Outpatient Clinic. IGF-1 (insulin-like growth factor 1) deficiency is one of the rarest causes of short stature. In 2009 in Poland a therapeutic programme was set up for children with severe primary IGF-1 deficiency. The authors present the data of three first polish patients qualified for the rhIGF-1 (recombinant human insulin-like growth factor 1) - mecasermin. The authors conclude that the treatment with rhIGF-1 significantly improves growth velocity in patients with IGF-1 deficiency. During two years of mecasermin treatment no serious side effects were noted.     

A phase II trial of temozolomide and IFN-alpha in patients with advanced renal cell carcinoma.

The combination of temozolomide (TEM) and interferon-alpha (IFN-alpha) previously demonstrated a 30% response rate in metastatic melanoma. A single institution, phase II trial evaluating the efficacy of TEM/IFN in patients with advanced renal cell carcinoma (RCC) was conducted. Safety and tumor response were the main outcomes. Eligible patients received 200 mg/m(2)/day TEM orally on days 1-5 every 28 days, with IFN 2.5 million U/m(2)/day subcutaneously (s.c.) three alternate days/week for days 1-15 first cycle, then 5 million U/m(2)/day s.c. 3 alternate days/week throughout each 28-day cycle. Efficacy was evaluated every 8 weeks, and dose-limiting toxicities (DLTs) were treated with dose reductions of the culprit drug. Sixteen patients (ages 37-67) were initially enrolled. Of the 14 evaluable patients, there was one minor response. Best response was stable disease, with 7 patients remaining on study for > or =6 months. Five were alive for more than 2 years, and 2 remain alive at 45 and 50 months after enrollment. DLTs included TEM-induced myelosuppression and IFN-induced fever/chills. Other toxicities were mild to moderate (grades 1-3). The combination of TEM/IFN proved quite tolerable. This regimen appears inactive in terms of response in this population with poor prognosis, but the patients with stable disease > or =6 months remain of interest.     

Distribution of HBV genotypes and mutants among hepatitis B infected patients from northern Poland

Hepatitis B virus (HBV) infection is one of the major global epidemiological problems. The aim of our study was to determine the distribution of HBV genotypes in Poland since the data concerning the spread of HBV viruses in the central-eastern region of Europe is still very limited. HBV DNA was extracted from 58 serum samples. To quantify the level of HBV DNA the Roche Amplicor HBV Monitor Assay was used. To genotype and assign HBV subtypes DNA sequencing methods were performed. The HBV virus from 43 serum samples from hepatitis B infected patients was genotype A (74.1%), 12 cases had genotype D (20.7%), and 3 had the rare in Europe genotype F (5.2%). Prediction of HBV serological subtypes based on HBsAg sequencing showed almost 100% occurrence of subtype adw2 in the group of genotype A samples, three different subtypes in genotype D (ayw2, ayw3, and ayw4), and equal distribution of subtype adw4q- in all 3 cases of genotype F, also the most prevalent subtype in the Amerindians. Our results coincide with the general European HBV prevalence. However, HBV genotype F, which is not a common genotype in European countries, was detected and so was relatively high occurrence of genotype D, which may reflect historical and ethnical migration events in Poland in the past.     

Latent autoimmune diabetes in adults in a population-based cohort of Polish patients with newly diagnosed diabetes mellitus

Introduction

Latent autoimmune diabetes in adults (LADA) is a slowly developing form of autoimmune diabetes characterized by the presence of type 1 diabetes-associated autoantibody. The aim of this study was to determine the incidence and characteristics of LADA in a population-based cohort of Polish patients with newly-diagnosed diabetes.

Material and methods

The study cohort was taken from the resident population of the city Białystok, Poland, during the period 1 January to 31 December 2003, aged 20-64 years. During this period we identified 231 cases of diabetes. We measured glutamic acid decarboxylase (GADA) and insulin antibody (IAA), insulin, C peptide and glycated hemoglobin (HbA_1c). Diagnosis of LADA was made according to Immunology Diabetes Society and Action LADA criteria.

Results

The incidence of LADA was 10 per year per 100 000 people. The proportion of patients with LADA was 8.9% among newly diagnosed cases with diabetes. Patients with LADA were younger at diagnosis (48.5 ±9.4 years vs. 54.8 ±10.6 years, p < 0.01), had lower body mass index (26.9 ±9.3 kg/m² vs. 29.5 ±5.2 kg/m², p < 0.05), C peptide (126 ±127 pmol/l vs. 446 ±592 pmol/l, p < 0.001), and were less insulin resistant (HOMA IR 0.94 ±0.85 vs. 3.6±4.4, p < 0.001) compared to patients with type 2 diabetes. Glycated hemoglobin and fasting glucose were similar in patients with LADA and type 2 diabetes.

Conclusions

In addition to GAD, anti-insulin antibodies are useful for diagnosing autoimmune diabetes in adults. Patients with LADA have similar glucose control parameters (HbA_1c) compared to patients with type 2 diabetes, although they are usually younger and have a lower body mass index. Patients with LADA make up a significant proportion of newly diagnosed people with diabetes mellitus in a Polish population.     

Effect of statins on platelet function in patients with hyperlipidemia

Introduction

It is generally assumed that cholesterol reduction by statins is the predominant therapeutic result underlying their beneficial effects in cardiovascular disease. However, the action of statins may be partially independent of their effects on plasma cholesterol levels, as they combine lipid lowering with positive effects on hemorheological conditions and endothelial function. We evaluated the impact of statin treatment on platelet adhesion to fibrinogen (spontaneous and ADP-activated), along with ADP, collagen or ristocetin-induced aggregation in type II hyperlipidemic patients.

Material and methods

The study group included 70 persons: 50 patients affected by type II hyperlipidemia without concomitant diseases and 20 healthy volunteers. The effects of 8-week statin treatment (atorvastatin 10 mg/day, simvastatin 20 mg/day, or pravastatin 20 mg/day) on platelet activation were evaluated.

Results

Regardless of the type of statin, a significant decrease in ADP-induced platelet aggregation was observed: for atorvastatin 50.6 ±12.8% vs. 41.1 ±15.8% (p < 0.05), for simvastatin 57.2 ±18.0% vs. 44.7 ±22.1% (p = 0.05), and for pravastatin 55.8 ±19.5% vs. 38.8 ±23.3% (p < 0.05). There was no significant effect of statins on collagen or ristocetin-induced platelet aggregation and adhesion.

Conclusions

Therapy with statins beneficially modifies ADP-induced platelet aggregation in patients with hyperlipidemia and does not affect spontaneous or ADP-induced platelet adhesion to fibrinogen and platelet aggregation induced by collagen or ristocetin.     

The role of intrahepatic lymphocytes in mediating protective immunity induced by attenuated Plasmodium berghei sporozoites

Summary: Exposure to irradiated Plasmodium sporozoites (g‐spz) results in protection against malaria. Like infectious spz, g‐spz colonize hepatocytes to undergo maturation. Disruption of liver stage development prevents the generation of protection, which appears, therefore, to depend on liver stage antigens. Although some mechanisms of protection have been identified, they do not include a role for intrahepatic mononuclear cells (IHMC). We demonstrated that P. berghei g‐spz‐immune murine IHMC adoptively transfer protection to naive recipients. Characterization of intrahepatic CD4+ T cells revealed an immediate, albeit transient, response to g‐spz, while the response of CD8+ T cells is delayed until acquisition of protection. It is presumed that activated CD8+ T cells home to the liver to die; g‐spz‐induced CD8+CD45RBloCD44hi T cells, however, persist in the liver, but not the spleen, during protracted protection. The association between CD8+CD45RBloCD44hi T cells and protection has been verified using MHC class I and CD1 knockout mice and mice with disrupted liver stage parasites. Based on kinetic studies, we propose that interferon‐g, presumably released by intrahepatic effector CD8+ T cells, mediates protection; the persistence of CD8+ T cells is, in turn, linked to Plasmodium antigen depots and cytokines released by CD4+ T cells and/or NK T cells.     

Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide

IntroductionInterleukin-2 (IL-2) when radiolabelled with ^99mTc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of ^99mTc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation.MethodsVarious molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The ^99mTc-labelling was optimized using various amounts of HYNIC–rhIL-2, ^99mTc, SnCl₂, tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats.ResultsGenerally, the highest radiolabelling yields were achieved when the HYNIC–rhIL-2 conjugates of ca. 2–4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of ^99mTc-HYNIC–rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the ^99mTc-HYNIC–rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 μg HYNIC–rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl₂. The monomer of ^99mTc-HYNIC–rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed.ConclusionsOur study shows that rhIL-2 can be efficiently radiolabelled with ^99mTc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use ^99mTc-HYNIC(tricine,NA)-rhIL-2 within 1 h.