In silico evaluation of limited sampling strategies for individualized dosing of extended half-life factor IX concentrates in hemophilia B patients.

PURPOSE: Hemophilia B is a bleeding disorder, caused by a factor IX (FIX) deficiency. Recently, FIX concentrates with extended half-life (EHL) have become available. Prophylactic dosing of EHL-FIX concentrates can be optimized by assessment of individual pharmacokinetic (PK) parameters. To determine these parameters, limited sampling strategies (LSSs) may be applied. The study aims to establish adequate LSSs for estimating individual PK parameters of EHL-FIX concentrates using in silico evaluation. METHODS: Monte Carlo simulations were performed to obtain FIX activity versus time profiles using published population PK models for N9-GP (Refixia), rFIXFc (Alprolix), and rIX-FP (Idelvion). Fourteen LSSs, containing three or four samples taken within 8 days after administration, were formulated. Bayesian analysis was applied to obtain estimates for clearance (CL), half-life (t1/2), time to 1% (Time1%), and calculated weekly dose (Dose1%). Bias and precision of these estimates were assessed to determine which LSS was adequate. RESULTS: For all PK parameters of N9-GP, rFIXFc and rIX-FP bias was generally acceptable (range: -5% to 5%). For N9-GP, precision of all parameters for all LSSs was acceptable (< 25%). For rFIXFc, precision was acceptable for CL and Time1%, except for t1/2 (range: 27.1% to 44.7%) and Dose1% (range: 12% to 29.4%). For rIX-FP, all LSSs showed acceptable bias and precision, except for Dose1% using LSS with the last sample taken on day 3 (LSS 6 and 10). CONCLUSION: Best performing LSSs were LSS with samples taken at days 1, 5, 7, and 8 (N9-GP and rFIXFc) and at days 1, 4, 6, and 8 (rIX-FP), respectively.

Secondary prevention of minor trauma fractures: the effects of a tailored intervention-an observational study.

INTRODUCTION: Minor trauma fractures (MTF) in the elderly are associated with an increase in mortality, morbidity, and the risk of subsequent fractures. Often, these patients who sustain MTF have an underlying bone disease, such as osteopenia or osteoporosis. Osteoporosis is known to be underdiagnosed and undertreated, and adequate treatment is essential to reduce the occurrence of MTFs. At our hospital, this has led to the implementation of Osteofit, a patient-education-based intervention targeted at improving screening and prevention of osteoporosis, with the goal to reduce the rate of subsequent MTF. OBJECTIVE: The aim of this study was to assess the efficacy of Osteofit in improving osteoporosis screening and treatment in patients after an initial MTF episode. METHODS: The study is a prospective, single-center, cohort study of MTF patients aged 50 years or older. A standardized questionnaire and telephone interview were used to collect 1-year follow-up data. The primary outcome was the rate of patients undergoing Dual X-ray Absorptiometry (DXA) scanning. Secondary outcomes were the rate of patients with a diagnosis of osteoporosis or osteopenia, the rate of patients treated with anti-osteoporotic medication, and the rate of patients with a subsequent fracture. DXA scanning rate, the prevalence of a diagnosis (osteoporosis/osteopenia), and data on medical treatment for osteoporosis were compared to the results of a previous study in the same hospital, published in 2004. RESULTS: Between 2012 and 2015, 411 of 823 eligible patients consented to participate and were included in this study. The mean age was 72 ± 9.3 years. Sixty-three percent (63.3%, n = 252) of the patients received a DXA scan, compared to 12.6% reported in our previous study. Of all patients who received a DXA scan, 199 (82.9%) were diagnosed with osteoporosis or osteopenia. A total of 95 patients (23.1%) received specific medical treatment for osteoporosis and 59.8% reported the intake of any unspecific medication (vitamin D, calcium, or both). Fifteen patients (3.9%) had a subsequent fracture as a result of a minor trauma fall. CONCLUSION: The implementation of a MTF secondary prevention program with dedicated health professionals improved the rate of patients who underwent DXA screening by fivefold. Despite this improvement, DXA screening was missed in over a third of patients, with only 23% of eligible patients receiving specific medical treatment for osteoporosis at 1-year follow-up. Consequently, this tailored intervention is a promising first step in improving geriatric fracture care. However, further work to improve the rate of osteoporosis screening and medical treatment initiation for the long-term prevention of subsequent MTF is recommended. We believe osteoporosis screening and adequate osteoporosis medication should be integrated as standard procedure in the aftercare of MTF. LEVEL OF EVIDENCE: II.

Lipid peroxidation affects red blood cells membrane properties in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune, chronic inflammatory, non-organ specific disease with an important morbimortality affecting several organs and systems. Oxidative stress is a well documented mechanism of red blood cells (RBC) mechanical impairment. Free radicals could produced, through lipid peroxidation, physical and chemical alterations in the cellular membrane properties modifying its composition, packing and lipid distribution on the membrane erythrocyte. The aim of the present work is to study the lipid peroxidation in the RBC membrane in SLE patients (n = 42) affecting so far the lipid membrane fluidity and erythrocyte deformability in comparison with healthy controls (n = 52). Malonildialdehyde (MDA) is a subrogate assessing lipidic peroxidation, rigidity index estimating erythrocyte deformability and the anisotropy coefficient estimating lipid membrane fluidity were used. Our results show that MDA values are increased, while erythrocyte deformability and membrane fluidity are significantly decreased in erythrocyte membrane from SLE patients in comparison with normal controls. The association of thiobarbituric acid reactive substances (TBARS) with membrane lipid fluidity and erythrocyte deformability confirms that the damage of membrane properties is produced by lipid peroxidation.

Environmental persistence of amphibian and reptilian ranaviruses.

Ranaviruses infect fish, amphibians, and reptiles. The present study was conducted to compare the persistence of amphibian and reptilian ranaviruses in a pond habitat. The 4 viruses used in this study included 2 amphibian ranaviruses, Frog virus 3 (FV3, the type species of the genus Ranavirus) and an isolate from a frog, and 2 ranaviruses of reptilian origin (from a tortoise and from a gecko). A sandwich germ-carrier technique was used to study the persistence of these viruses in sterile and unsterile pond water (PW) and soil obtained from the bank of a pond. For each virus, virus-loaded carriers were placed in each of the 3 substrates, incubated at 4 and 20°C, and titrated at regular intervals. Serial data were analyzed using a linear regression model to calculate T-90 values (time required for 90% reduction in the virus titer). Resistance of the viruses to drying was also studied. All 4 viruses were resistant to drying. At 20°C, T-90 values of the viruses were 22 to 31 d in sterile PW and 22 to 34 d in unsterile PW. Inactivation of all 4 viruses in soil at this temperature appeared to be non-linear. T-90 values at 4°C were 102 to 182 d in sterile PW, 58 to 72 d in unsterile PW, and 30 to 48 d in soil. Viral persistence was highest in the sterile PW, followed by the unsterile PW, and was lowest in soil. There were no significant differences in the survival times between the amphibian and reptilian viruses. The results of the present study suggest that ranaviruses can survive for long periods of time in pond habitats at low temperatures.

Erythrocyte aggregation in patients with systemic lupus erythematosus.

Systemic Lupus Erythematosus (SLE) is an autoimmune, chronic inflammatory, non-organ specific disease. SLE patients present a high prevalence of thrombotic and arteriosclerotic disease. The aim of the present work was to study the erythrocyte aggregation kinetics, and the effect of plasma factors, namely, immunoglobulin and fibrinogen concentration, as well as cell factors such as deformability and erythrocyte membrane lipid fluidity on the erythrocyte aggregation, in SLE patients and healthy controls. The results show that SLE patients red blood cells aggregate at higher rate and the aggregates size are also greater than controls due to an increase of immunoglobulin and plasma fibrinogen. The negative correlation between aggregation parameters and rigidity index could point out that the altered deformability diminishes the erythrocyte aggregation. Correlation between rigidity index and anisotropy suggests that the decrease of membrane lipid fluidity might be a cause of deformability decrease. The erythrocyte aggregation increase in these patients could induce a decreased flow that might contribute to the thromboembolic process present in SLE patients.

Effects of ascorbate fatty ester derivatives on erythrocyte membrane lipoperoxidation.

6-O-alkyl ascorbic acid esters (ASC(n)) are amphiphilic molecules that behave as surfactants in aqueous solution. ASC(n) have shown some physical and rheological properties that suggest a potential utility as drug carriers. The present paper aims to evaluate the effect of ASC(n) on erythrocyte properties in order to get information regarding the relationship between osmotic fragility, erythrocyte deformability and membrane lipoperoxidation process. The assays were performed at the following concentrations: the critical micelar concentration (CMC), producing 10% hemolysis (CH(10)) and producing 50% hemolysis (CH(50)). We observed that ASC(n) (ASC(8), ASC(10) and ASC(12)), at concentration nearby CMC, neither affected cell deformability nor produced lipoperoxidation. Nevertheless, at higher concentrations (CH(10) and CH(50)), the RBCs incubated with ASC(n) were affected by a significant and progressive loss of deformability, simultaneously with an increase of osmotic fragility and membrane lipoperoxidation.

Study on membrane fluidity and erythrocyte aggregation in equine, bovine and human species.

The aim of the present paper is to analyze whether membrane fluidity can be predicted from its lipid composition and to assay the possible relationship between such variable and the aggregating properties of erythrocytes from equine, bovine and human species due to the widely acknowledged differences in their tendency to form aggregates. The main difference between phospholipids from plasma membrane in these species lies in the concentration levels of sphyngomyelin (SM) and phosphatidilcoline (PC); more precisely, in the external hemilayer of the lipid bilayer. Membrane fluidity was estimated by the fluorescence polarization method, while erythrocyte aggregation was assessed by an optical method. According to our results, bovine erythrocytes containing high SM and low PC levels, presented the highest anisotropy value as well as an imperceptible aggregation value. Equine erythrocytes, which contain a considerable PC percentage and scarce SM levels, showed the lowest anisotropy value and the highest values of the aggregation parameters. Human erythrocytes presented intermediate values for both properties. Our hypothesis claims that the phospholipid composition would constitute one of the factors determining erythrocyte membrane fluidity and also taking part in the different aggregation tendency shown by equine, bovine and human species.

Nifedipine effect on red cell rheological properties in patients with systemic scleroderma.

Systemic scleroderma is an autoimmune disease, due to a connective tissue alteration characterized by extracellular matrix increase in the skin and internal organs. It is already known that the Raynaud's phenomenon and the microcapillary obliteration lead to ischemia and peripheral tissue injury. The ischemia-reperfusion phenomenon releases free radicals, that react with red blood cells (RBCs) membrane components originating lipid peroxidation and impairment of the ATP-Ca(++) pump, two possible mechanisms responsible of disease pathogenesis. Nifedipine is a Ca(++)-channel antagonist that has been used for a long time in Raynaud's phenomenon treatment. In the present study we were able to demonstrate that erythrocyte deformability and two other related variables such as membrane fluidity and osmotic fragility improve significantly with nifedipine therapy. It is likely that nifedipine inhibiting cytoplasmic calcium accumulation could restore some red blood cell membrane properties.

Effect of ascorbic acid based amphiphiles on human erythrocytes membrane.

6-O-alkyl ascorbic acid esters (ASCn) are amphiphilic molecules that behave as surfactants in aqueous solution. These compounds show physico-chemical and aggregation properties that depend on the alkyl chain length, pH and temperature. It must consider that ASCn have shown some physical and rheological properties that suggest a potential utility as drug carriers. The present paper aims to evaluate the effects of these surfactants on human erythrocyte membranes. The membrane properties studied were: osmotic resistance in hypotonic media, shape transformation, and vesicle release at lytic concentration. According to our results, all properties depended on the length of the hydrophobic chain and they did not evolve monotonically. Finally, the study of ASCn interaction with erythrocyte membrane allowed us to postulate the crucial influence that the molecular structure exerts upon the manner in which amphiphiles interact with biological membranes and the effects involved in them.

Evaluation of the efficacy and tolerability of acarbose in patients with diabetes mellitus : a postmarketing surveillance study.

OBJECTIVE: The efficacy and tolerability of acarbose were examined in a postmarketing surveillance study of 27 803 patients with diabetes mellitus (26 044 were diagnosed as having type 2 diabetes) over a 12-week treatment period. PATIENTS AND M ethods: Overall efficacy data were reported for type 1 and type 2 diabetes, and a detailed data analysis was conducted for patients with type 2 diabetes. Tolerability was described for the total group. Of the type 2 diabetes patients, 37.6% were treated with diet only; 44.2% were additionally treated with sulphonylureas; 6.3% with metformin or metformin plus sulphonylurea; and 11.6% with insulin alone or in combination with oral treatment. The frequency of two or more concomitant diseases was 45.8% for all type 2 diabetes patients, and 62.4% in elderly patients (age >/=70 years). RESULTS: In patients with type 2 diabetes, acarbose administration in addition to the existing treatment resulted in reductions in mean blood glucose levels (fasting 50 mg/dL, 1h post-prandial [pp] 60 mg/dL, 2h pp 56 mg/dL), glycosylated haemoglobin (HbA(1c) 1.3%; HbA(1) 1.6%) and bodyweight (1.5 kg). Results for type 1 diabetes patients were similar. No clinically relevant influence of age, body mass index or number of concomitant diseases on the results could be observed. Tolerability was good: 83% of patients had no adverse events, 13.7% reported flatulence, and 2.2% had at least one occurrence of diarrhoea. Hypoglycaemia was found in 0.07% of patients, mainly in combination with metformin or insulin. Tolerability was independent of patients' age. Laboratory investigations gave no indication of other adverse events. CONCLUSION: This postmarketing surveillance study documents the therapeutic benefit and good tolerability and compliance of acarbose as mono- and combination therapy, even in elderly and multimorbid patients.

Association between capillaroscopy, haemorheological variables and plasma proteins in patients bearing Raynaud's phenomenon.

Raynaud's phenomenon is a paroxysmal and reversible vasospasm affecting generally the acral circulatory regions. The relevance of the haemorheological alterations in these patients, as a source of ischemic events has been neglected. The objective of the present work was to evaluate and correlate the rheological blood properties, some biochemical parameters, e.g., plasma fibrinogen and immunoglobulin levels, and periungual capillaroscopy. The explicative variables considered were: blood viscosity, plasma viscosity, erythrocyte rigidity index, plasma fibrinogen, erythrocyte sedimentation rate, erythrocyte aggregate size, erythrocyte aggregation rate and serum immunoglobulin (IgG and IgM). The response variable was the nailfold capillary pattern categorised as either normal or pathological. Fibrinogen, erythrocyte aggregation rate and IgM are significantly higher in patients with a pathological pattern in comparison with patients bearing a normal one. The statistical analysis enabled us the modelling of the pathological pattern occurrence probability in function of plasma fibrinogen. Consequently, 100 mg/dl plasma fibrinogen increase, increases twice the probability of presenting a pathological pattern. Therefore, we can conclude that high levels of fibrinogen in Raynaud's phenomenon patients are associated with impaired skin microcirculation assessed by periungual capillaroscopy.

Effect of hormone replacement therapy upon haemorheological variables.

It is already admitted that hormone replacement therapy (HRT) decreases the risk of developing cardiovascular disease, although its mechanism is not clear yet. In the present work, the effect of the HRT upon cellular and plasmatic haemorheological factors determining blood flow properties: blood viscosity, plasma viscosity, plasma fibrinogen, rigidity erythrocyte index and erythrocyte aggregation rate was studied. Menopausal women were followed through a whole year of HTR. Results demonstrate that after six months of treatment there is a diminution in relative blood viscosity and erythrocyte rigidity, with constant values along the second semester. Erythrocyte aggregation, plasmatic and blood viscosity diminution observed during the treatment can be explained by the simultaneous plasma fibrinogen decrease. Modified cellular and plasmatic rheology could produce beneficial effects on blood flow, particularly in microcirculation, presenting a possible mechanism by which HTR decreases the risk of cardiovascular disease development during menopause.

Influence of plasma proteins on erythrocyte aggregation in three mammalian species.

The aggregation capacity of human erythrocytes lies between that of the non-aggregating bovine erythrocytes and the remarkably aggregating equine ones. As the ability to aggregate is attributed to cell factors and the composition of the plasma proteins, the role that plasma proteins play in the aggregation process in these three species was studied. Washed erythrocytes were suspended in phosphate-buffered saline (PBS; pH 7.4, 300 mOsm/L) plus polyvinylpyrrolidone (PVP) in a suitable concentration to obtain an average intensity of aggregation (control media). The superimposed effect of replacing 80% of the medium by either autologous plasma, serum or albumin solution was studied. The plasma proteins appeared to enhance aggregation by human and equine erythrocytes, but impaired this process in bovine erythrocytes. Some evidence was obtained supporting the existence of serum factors capable of reducing aggregation of erythrocytes in cattle and it was concluded that the non-aggregating behaviour of bovine erythrocytes may be due to the cells interacting particularly with the macromolecules in the serum.

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.

Interactions among structural proteins of varicella zoster virus.

Varicella zoster virus tegument components include the regulatory proteins IE4, IE62, IE63 and the ORFI0 protein, a protein kinase (ORF47) and an abundant protein encoded in ORF9 which is the homolog of HSV VP22. The kinase is able to phosphorylate IE62 and the ORF9 protein specifically in viral particles. We show that interactions among these proteins are, at least in part, dependent on the presence or absence of phosphate groups and we suggest models for tegument formation and for its dissolution in the infected cell.

Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development.

We report loss- and gain-of-function analyses that identify essential roles in development for Drosophila transcription factor AP-2. A mutagenesis screen yielded 16 lethal point mutant alleles of dAP-2. Null mutants die as adults or late pupae with a reduced proboscis, severely shortened legs (approximately 30% of normal length) lacking tarsal joints, and disruptions in the protocerebral central complex, a brain region critical for locomotion. Seven hypomorphic alleles constitute a phenotypic series yielding hemizygous adults with legs ranging from 40-95% of normal length. Hypomorphic alleles show additive effects with respect to leg length and viability; and several heteroallelic lines were established. Heteroallelic adults have moderately penetrant defects that include necrotic leg joints and ectopic growths (sometimes supernumerary antennae) invading medial eye territory. Several dAP-2 alleles with DNA binding domain missense mutations are null in hemizygotes but have dominant negative effects when paired with hypomorphic alleles. In wild-type leg primordia, dAP-2 is restricted to presumptive joints. Ectopic dAP-2 in leg discs can inhibit but not enhance leg elongation indicating that functions of dAP-2 in leg outgrowth are region restricted. In wing discs, ectopic dAP-2 cell autonomously transforms presumptive wing vein epithelium to ectopic sensory bristles, consistent with an instructive role in sensory organ development. These findings reveal multiple functions for dAP-2 during morphogenesis of feeding and locomotor appendages and their neural circuitry, and provide a new paradigm for understanding AP-2 family transcription factors.

Mushroom bodies and post-mating behaviors of Drosophila melanogaster females.

After mating, Drosophila melanogaster females lay substantially more eggs and mate rarely. Central to these changes is Sex peptide (SP), a male peptide transferred into the female during copulation. Injected into virgins, SP induces the same post mating response as observed after mating. In this study we investigated the role of the mushroom body (MB) in the SP response system. The SP response of females with either chemically ablated or mutant MBs was analyzed. After injection of SP, females with chemically ablated MBs reduce their receptivity and increase their ovulation and oviposition to the level of females with intact MBs. Virgin females with ablated MBs, however, show a constitutively elevated oviposition rate. Hence in untreated females, MBs are not implicated in the SP-induced reduction of receptivity and increase of ovulation. However, they depress the oviposition rate of virgins. Thus, SP has two functions for oviposition: it de-represses the MB-dependent block on the egg laying activity of virgins and additionally stimulates oviposition. SP-injected mushroom body miniature (mbm) females lay fewer eggs, ovulate less frequently, and mate more often than wild-type females. A model of the putative role of MBs and the gene product of mbm in SP-induced oviposition is presented.

Physical interaction between two varicella zoster virus gene regulatory proteins, IE4 and IE62.

Transfection assays demonstrate that the varicella zoster virus (VZV) immediate-early 62 (IE62) protein is a major transactivator of VZV gene expression, whereas a second immediate-early protein, IE4, can act as a major coactivator of transactivation mediated through IE62. To test whether IE62 and IE4 interact physically, we performed several protein-protein interaction assays. Coimmunoprecipitation analyses using VZV-infected cell lysates as well as purified protein mixtures demonstrate that IE62 and IE4 form stable complexes in solution under stringent salt conditions. Enzyme-linked immunosorbent assay protein-protein interaction assays and maltose-binding protein capture assays demonstrate that IE62 binds IE4 in a concentration- and dose-dependent manner. Far Western blot analyses show that IE4 binds to an undermodified form of IE62, and the use of calf intestinal phosphatase and protein kinases suggests that the interaction with IE4 is dependent on the phosphorylation state of IE62. An IE4 binding domain on IE62 has been mapped using a set of truncated IE62 fusion peptides. Collectively, these results imply a direct and specific physical interaction between IE4 and less-phosphorylated forms of IE62. These data have implications for virion assembly, as well as for the regulation of gene expression in VZV-infected cells.

Acarbose treatment does not change the habitual diet of patients with Type 2 diabetes mellitus. The Finnish Acargbos Study Group.

AIMS: It has been speculated that acarbose treatment in patients with Type 2 diabetes mellitus might induce changes in diet as a result of its adverse gastrointestinal effects. The aim of this study was to determine whether poor metabolic control can be improved by acarbose, and whether this might be because the acarbose supplementation provokes changes in diet. METHODS: Poorly controlled Type 2 diabetic patients treated with oral hypoglycaemic agents (OHA) were randomized into either acarbose (100 mg t.d.s.) or placebo treatment. The double-blind treatment lasted for 24 weeks. Four-day food diaries and blood samples for efficacy analysis were collected at 0, 4, 12, and 24 weeks. Thirty-six acarbose and 39 placebo-treated patients completed the trial and were included in the final analyses. RESULTS: At 24 weeks the baseline adjusted means of fasting, 1 and 2-h postprandial blood glucose values were 9.3 vs. 10.5 (P=0.02), 11.6 vs. 14.5 (P<0.001) and 11.0 vs. 13.7 mmol/l (P<0.001) and HbA1 9.3% vs. 10.2% (P=0.002) in the acarbose and placebo groups, respectively. No significant differences in nutrient intakes between groups were observed. The energy intake and energy proportion of fat and carbohydrates remained unchanged in both groups. CONCLUSIONS: Acarbose significantly improves metabolic control in Type 2 diabetic patients poorly controlled with oral hypoglycaemic agents. This effect seems not to be a result of concomitant involuntary dietary changes, since acarbose did not induce modifications in diet during the study.