In silico and in vitro immunogenicity assessment of B-domain-modified recombinant factor VIII molecules.

INTRODUCTION: B-domain modification can improve production of recombinant factor VIII (rFVIII) proteins. However, the engineered junction results in non-native peptide sequences with the potential to elicit immune responses via major histocompatibility complex class-II (MHC-II)-binding and CD4+ T cell activation. AIM: Assess the immunogenic potential of B-domain junction peptides of turoctocog alfa and other B-domain-modified rFVIII proteins using in silico and in vitro immunogenicity assessment techniques. METHODS: Peptides with amino acid sequences identical to the B-domain junction of turoctocog alfa, simoctocog alfa and moroctocog alfa were evaluated by in silico peptide-MHC-II binding prediction, in vitro peptide-MHC-II-binding measurement and in vitro T cell-activation assays. Moreover, turoctocog alfa was assessed for peptide presentation on dendritic cells (DCs) using MHC-associated peptide proteomics. RESULTS: In silico analysis predicted virtually no neo-epitopes in the B-domain junction for turoctocog alfa, whereas some were predicted for simoctocog alfa and moroctocog alfa. Turoctocog alfa and moroctocog alfa peptides showed minimal capacity to bind high-frequency MHC-II molecules in vitro, whereas simoctocog alfa peptide demonstrated some degree of binding to approximately half of the MHC-II molecules tested. In line with this, no B-domain peptides from turoctocog alfa were found to be presented on MHC-II complexes on DCs. B-domain junction peptides from all 3 compounds induced T cell responses in only a few percentages of donors. CONCLUSION: All 3 junction peptides were found to have a low immunogenicity potential, suggesting that modification of the B-domain does not constitute an increased immunogenicity risk for any of the products examined.

[Snake bite in a 53-year-old female tourist]. / 53-jährige Urlauberin auf Hiddensee mit Schlangenbiss.

Snake bites are rare events in Germany and are not life-threatening with usually only mild clinical symptoms. The most widespread venomous snake is the common European adder (Vipera berus). Here we present the case of a 53-year-old woman who was bitten by a common adder. Although the patient was initially in stable condition she developed edematous swelling of the complete lower limb, subcutaneous bleeding, and rhabdomyolysis. The aim of this report is to raise awareness that even in a central European country like Germany snake bites with a life-threatening course can occur and need immediate attention and medical care.

[Maxillary ameloblastoma extending into the sinonasal tract]. / Expansion eines Ameloblastoms in den sinunasalen Trakt.

Maxillary ameloblastomas can extensively expand into the paranasal sinuses or even the nasal cavity due to a slow growth pattern. Sinusitis is rarely the first tumor-related complaint. Due to the various growth forms of ameloblastomas the challenging histological differential diagnosis includes several other odontogenic as well as benign and malignant non-odontogenic tumors, e.g. tumors from the mucosa of the paranasal sinuses, salivary glands and Rathke's pouch. Despite the radical surgical approach a complete resection with wide margins cannot always be achieved. Maxillary ameloblastomas show the highest recurrence rates.

Pituitary choriocarcinoma in an adolescent male: tumor-derived CG and GH delay diagnosis.

BACKGROUND: Primary intracranial germ cell tumors usually present in the first two decades of life, often with precocious puberty. The most common location is in the pineal gland; suprasellar germ cell tumors are rare. We present an additional case of a suprasellar choriocarcinoma producing GH, and review the literature. CASE: This French Canadian, 17 year-old male presented to the ER with a history of mild weight loss and an episode of syncope while hiking in Mexico, but with no other neurological symptoms. Puberty began at age 13 years (growth spurt: 15-16 years), and he attained an adult height within genetic target by age 16 years. Past medical history was negative except for myopia diagnosed during childhood. System review revealed increased thirst and nocturia. The mother was treated for an oligo-astrocytoma in 2007. Clinical examination showed a euthyroid, well-looking young man with 20 ml testicles. Endocrine evaluation revealed elevated testosterone, mildly elevated PRL, borderline low FT4, and decreased IGF-I, morning cortisol and urine osmolality; tumor markers were positive in serum and CSF (hCG>50 IU/L, AFP>10 ng/mL). A transphenoidal biopsy of a 4.5 cm, homogeneous, non-calcified, suprasellar mass was compatible with the diagnosis of choriocarcinoma and stained intensely for hCG and hGH, presumably the placental variant (GH-V) as previously found in vitro in choriocarcinoma cell lines. Combined chemotherapy and irradiation led to tumor regression and undetectable serum hCG to 36 months of follow-up. He is doing well with no evidence of tumor progression and is on complete hormone replacement therapy. CONCLUSIONS: Choriocarcinomas can have a hormonal profile that delays the development of symptoms, due to hCG stimulation of both the gonadal and thyroid axes. This report corroborates previous in vitro evidence that choriocarcinoma cells are able to make GH-V. To what extent the patient's tumor-derived GH contributed to his normal growth is not known. Prognosis for this intracranial neoplasm is very reserved, although combined radiotherapy and chemotherapy has been successful in our patient now 36 months post-diagnosis.

[Restoration and maintenance of sinus rhythm in patients with atrial fibrillation. New pharmacological approaches]. / Wiederherstellung und Aufrechterhaltung eines Sinusrhythmus bei Patienten mit Vorhofflimmern. Neue medikamentöse Therapieansätze.

Atrial fibrillation is the most frequent cardiac arrthythmia in adults and its prevalence is increasing with age. Therefore, the importance of an adequate therapy is an increasing challenge, in particular considering the demographic shift towards an aging population. Current antiarrhythmic drug therapies for the conversion of atrial fibrillation and the maintenance of sinus rhythm are limited by efficacy, tolerance and safety of the currently available agents. Therefore, a primary goal is to develop effective antiarrhythmic drugs with as little as possible side effects. The following review describes new promising drug therapy approaches to atrial fibrillation that may overcome some of the limitations of current therapies.

Prevalence of risk determinants for metformin-associated lactic acidosis and metformin utilization in the study of health in pomerania.

Risk determinants for the life threatening complication of metformin-associated lactic acidosis are frequently disregarded. Our first aim was to investigate the prevalence of risk determinants in subjects with type 2 diabetes mellitus (DM2) taking metformin compared to subjects with nonmetformin treatment. Our second aim was to estimate the proportion of subjects with alternative drug-treatment, and no risk determinants, which would probably benefit from metformin. The Study of Health in Pomerania is a population-based health survey including 322 DM2 subjects. Risk determinants were assessed by personal interview, ultrasound, and laboratory analysis. Among the subjects with DM2 n=92 (28.6%) were treated with metformin, n=162 (50.3%) with alternative medication, and n=68 (21.1%) with diet. The prevalence of at least one risk determinant was 65% [corrected] for metformin-users. There was no difference in number and type of risk determinants. Heart failure, angina pectoris, and liver disorders presented the most frequent risk determinants. Current risk determinants for metformin-associated lactic acidosis are largely disregarded. Improved selection of patients can result in safe metformin utilization in one quarter of subjects on DM2 related drug treatment. Risk determinants need to be revised. A more practical definition of risk determinants would improve prescription adherence.

Metformin-associated lactic acidosis in patients with renal impairment solely due to drug accumulation?

AIM: We suspect that the life-threatening complication of metformin-associated lactic acidosis, solely due to drug accumulation following renal impairment, occurs more frequently than that previously reported and is not necessarily associated with other predisposing factors for lactic acidosis. METHODS: During a period of 13 months, at a tertiary referral centre, the incidence of lactic acidosis of any aetiology was 12.8% [67 of 524 total intensive care unit (ICU) admissions]. Metformin-associated lactic acidosis solely as the result of drug accumulation was diagnosed in 6% of all the patients suffering from lactic acidosis (4 of 67 patients). RESULTS: These patients presented with severe circulatory shock due to lactic acidosis. We could not identify any predisposing factor for lactic acidosis other than renal impairment. Intercurrent deterioration of diabetic nephropathy was suspected to be responsible for the accumulation of metformin followed by lactic acidosis, finally resulting in multiorgan failure. The diagnosis was supported by extensively elevated serum levels of metformin. Two patients died during ICU treatment. CONCLUSIONS: Our data indicate that the incidence of metformin-associated lactic acidosis solely due to metformin accumulation is possible and underestimated. Symptoms of metformin-associated lactic acidosis are unspecific and physicians should be aware that metformin, if prescribed in patients with renal impairment, can cause fatal lactic acidosis due to drug accumulation.

Cyclosporine-loaded solid lipid nanoparticles (SLN): drug-lipid physicochemical interactions and characterization of drug incorporation.

Solid lipid nanoparticles (SLN) were produced loaded with cyclosporine A in order to develop an improved oral formulation. In this study, the particles were characterized with regard to the structure of the lipid particle matrix, being a determining factor for mode of drug incorporation and drug release. Differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS) measurements were employed for the analysis of the polymorphic modifications and mode of drug incorporation. Particles were produced using Imwitor 900 as lipid matrix (the suspension consisted of 10% particles, 8% Imwitor 900, 2% cyclosporine A), 2.5% Tagat S, 0.5% sodium cholate and 87% water. DSC and WAXS were used to analyse bulk lipid, bulk drug, drug incorporated in the bulk and unloaded and drug-loaded SLN dispersions. The processing of the bulk lipid into nanoparticles was accompanied by a polymorphic transformation from the beta to the alpha-modification. After production, the drug-free SLN dispersions converted back to beta-modification, while the drug-loaded SLN stayed primarily in alpha-modification. After incorporation of cyclosporine A into SLN, the peptide lost its crystalline character. Based on WAXS data, it could be concluded that cyclosporine is molecularly dispersed in between the fatty acid chains of the liquid-crystalline alpha-modification fraction of the loaded SLN.

Oral bioavailability of cyclosporine: solid lipid nanoparticles (SLN) versus drug nanocrystals.

For the development of an optimized oral formulation for cyclosporine A, 2% of this drug has been formulated in solid lipid nanoparticles (SLN, mean size 157 nm) and as nanocrystals (mean size 962 nm). The encapsulation rate of SLN was found to be 96.1%. Nanocrystals are composed of 100% of drug. For the assessment of the pharmacokinetic parameters the developed formulations have been administered via oral route to three young pigs. Comparison studies with a commercial Sandimmun Neoral/Optoral used as reference have been performed. The blood profiles observed after oral administration of the commercial microemulsion Sandimmun revealed a fast absorption of drug leading to the observation of a plasma peak above 1,000 ng/ml within the first 2 h. For drug nanocrystals most of the blood concentrations were in the range between 30 and 70 ng/ml over a period of 14 h. These values were very low, showing huge differences between the measuring time points and between the tested animals. On the contrary, administration of cyclosporine-loaded SLN led to a mean plasma profile with almost similarly low variations in comparison to the reference microemulsion, however with no initial blood peak as observed with the Sandimmun Neoral/Optoral. Comparing the area under the curves (AUC) obtained with the tested animals it could be stated that the SLN formulation avoids side effects by lacking blood concentrations higher than 1,000 ng/ml. In this study it has been proved that using SLN as a drug carrier for oral administration of cyclosporine A a low variation in bioavailability of the drug and simultaneously avoiding the plasma peak typical of the first Sandimmun formulation can be achieved.

Alanine-stimulated exocytosis in Aplysia enterocytes: effect of Na+ transport and requirement for actin filaments.

We used the Aplysia californica intestinal epithelium to investigate the effect of alanine-stimulated Na+ absorption on apical membrane exocytosis and whether stimulated exocytosis requires intact actin filaments. The fluid-phase marker fluorescein dextran was used to determine rates of apical membrane exocytosis. L-alanine significantly increased apical exocytosis by approximately 30% compared to controls, and there is a modest, positive correlation between alanine-stimulated exocytosis and short-circuit current (ISC). Thus, apical exocytosis is modulated to some extent by the magnitude of Na+ and alanine entry across the apical membrane. Apical exocytosis is also responsive to virtually any increase in Na+ and alanine entry because increments in alanine-stimulated ISC as small as 1 microA/cm2 stimulated exocytosis. We used D-alanine to determine which parameter (sensitivity to transport vs. magnitude of transport) was most important in activation of apical exocytosis. D-alanine-stimulated ISC was one-sixth that of L-alanine, but stimulated exocytosis was only 29% less than that of L-alanine. Therefore, the apical exocytic system is more responsive to small increases in transport than to the magnitude of transport. Latrunculin A (Lat-A) disrupts the actin cytoskeleton and reduced constitutive apical exocytosis by approximately 65% and completely abolished alanine-stimulated exocytosis. Hence, constitutive exocytosis and alanine-stimulated exocytosis require actin filaments for recruitment of vesicles to the apical membrane. During nutrient absorption, actin filament-regulated apical exocytosis may represent a negative feedback system that modulates apical membrane tension.

Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity.

Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.

Different domains of the glucagon and glucagon-like peptide-1 receptors provide the critical determinants of ligand selectivity.

(1) Glucagon and glucagon-like peptide-1 (GLP-1) are homologous peptide hormones with important functions in glucose metabolism. The receptors for glucagon and GLP-1 are homologous family B G-protein coupled receptors. The GLP-1 receptor amino-terminal extracellular domain is a major determinant of glucagon/GLP-1 selectivity of the GLP-1 receptor. However, the divergent residues in glucagon and GLP-1 that determine specificity for the GLP-1 receptor amino-terminal extracellular domain are not known. Less is known about how the glucagon receptor distinguishes between glucagon and GLP-1. (2) We analysed chimeric glucagon/GLP-1 peptides for their ability to bind and activate the glucagon receptor, the GLP-1 receptor and chimeric glucagon/GLP-1 receptors. The chimeric peptide GLP-1(7-20)/glucagon(15-29) was unable to bind and activate the glucagon receptor. Substituting the glucagon receptor core domain with the GLP-1 receptor core domain (chimera A) completely rescued the affinity and potency of GLP-1(7-20)/glucagon(15-29) without compromising the affinity and potency of glucagon. Substituting transmembrane segment 1 (TM1), TM6, TM7, the third extracellular loop and the intracellular carboxy-terminus of chimera A with the corresponding glucagon receptor segments re-established the ability to distinguish GLP-1(7-20)/glucagon(15-29) from glucagon. Corroborant results were obtained with the opposite chimeric peptide glucagon(1-14)/GLP-1(21-37). (3) The results suggest that the glucagon and GLP-1 receptor amino-terminal extracellular domains determine specificity for the divergent residues in the glucagon and GLP-1 carboxy-terminals respectively. The GLP-1 receptor core domain is not a critical determinant of glucagon/GLP-1 selectivity. Conversely, the glucagon receptor core domain contains two or more sub-segments which strongly determine specificity for divergent residues in the glucagon amino-terminus.

In situ hybridization.

In situ hybridization (ISH) is employed principally to detect mRNA within cells and tissues. ISH begins with the synthesis of a nucleic acid probe complementary in sequence to a cellular target. Through incorporation of a radioactive nucleotide into the probe, a cellular target may be visualized using autoradiography. A nonradioactive variation of this technique utilizing digoxigenin has been championed by some researchers, but our experience has been that radioactive probes produce more reliable and accurate results. Although not a difficult technique, ISH incorporates many steps, which must be performed with care and precision to obtain useful results.

H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein.

H19 RNA is a major oncofetal 2.5-kilobase untranslated RNA of unknown function. The maternally expressed H19 gene is located 90 kilobase pairs downstream from the paternally expressed insulin-like growth factor II (IGF-II) gene on human chromosome 11 and mouse chromosome 7; and due to their reciprocal imprinting and identical spatiotemporal expression, it is assumed that the two genes are functionally coupled. Here we show that human H19 RNA contains four attachment sites for the oncofetal IGF-II mRNA-binding protein (IMP) with apparent K(d) values in the 0.4-1.3 nm range. The multiple attachment sites are clustered within a 700-nucleotide segment encoded by exons 4 and 5. This 3'-terminal segment targets H19 RNA to lamellipodia and perinuclear regions in dispersed fibroblasts where IMP is also localized. The results suggest that IMP participates in H19 RNA localization and provides a link between the IGF-II and H19 genes at post-transcriptional events during mammalian development.

Queuing for coronary angiography during severe supply-demand mismatch in a US public hospital: analysis of a waiting list registry.

CONTEXT: Adverse cardiac events have been reported in patients waiting for either coronary surgery or angioplasty. However, data on the risk of adverse events while awaiting coronary angiography are limited, and none are available from a US population. OBJECTIVE: To quantify cardiac outcomes in patients waiting for elective coronary angiography. DESIGN, SETTING, AND PARTICIPANTS: Observational cohort study of 381 adult outpatients (mean [SD] age, 55 [12] years; 64% male; 61% white) on a waiting list for coronary angiography at a US tertiary care public teaching hospital during 1993-1994. MAIN OUTCOME MEASURES: Rates of cardiac death, nonfatal myocardial infarction, and hospitalizations for unstable angina or heart failure as a function of amount of time spent on a waiting list. RESULTS: Sixty-six patients were dropped from the waiting list but were included in the study analysis. During a mean (SD) follow-up of 8.4 (6.5) months, cardiac death, myocardial infarction, and hospitalization occurred in 6 (1.6%), 4 (1.0%), and 26 (6.8%) patients, respectively. The probability of events was minimal in the first 2 weeks and increased steadily between 3 and 13 weeks. By Cox multivariate analysis, 2 variables independently identified an increased risk of adverse events: a strongly positive treadmill exercise electrocardiogram or positive stress imaging result at referral (odds ratio [OR], 2.32; 95% confidence interval [CI], 1.22-4.16; P=.01) and the use of 2 to 3 anti-ischemic medications (OR, 1.98; 95% CI, 1.19-3.96; P=.04). Among 311 patients who ultimately underwent angiography, those with adverse events had a higher prevalence of coronary disease (96% vs 60%; P<.001), more frequently required revascularization (93% vs 53%; P<.001), and had longer hospital stays (mean [SD], 6.2 [4.3] vs 1.3 [0.7] days; P=.001). CONCLUSION: Our data suggest that in a cohort referred for coronary angiography, delaying the procedure places some patients at risk for death, myocardial infarction, unplanned hospitalization, a longer hospital stay, and, potentially, a poorer prognosis. Waits longer than 2 weeks should be avoided, and patients with strongly positive stress test results and those who require 2 to 3 anti-ischemic medications should be prioritized for early intervention.

A simple, inexpensive method for teaching how membrane potentials are generated.

We have developed a simple laboratory exercise that uses an inexpensive dialysis membrane (molecular weight cutoff = 100) to illustrate the generation of membrane potentials (Vm) across plasma membranes of animal cells. A piece of membrane approximately 2.0 cm2 is mounted in an Ussing-like chamber. One chamber half is designated cytosol and the other half external. Chamber sidedness helps students relate their findings to those of real cells. As in real cells, outward directed K+ concentration gradients [high cytosolic K+ concentration ([K+]c) and low extracellular K+ concentration] generate cytosol electrically negative Vm with a slope of approximately -45 mV/decade change in [K+]c. The polarity of Vm reflects the outward flow of potassium ions because flow of the larger counterion, H2PO4-, is restricted to the pores in the membrane. A slope less than Nernstian (<59 mV/decade) suggests that the membrane is slightly permeable to H2PO4-. Importantly, this facilitates teaching the use of the Nernst equation to quantify the relationship between ion concentration ratios across membranes and magnitude of Vm. For example, students use their data and calculate a permeability ratio PK/PH2PO4 that corresponds to a slope of approximately 24% less than Nernstian. This calculation shows that Nernstian slopes are achieved only when permeability to the counterion is zero. Finally, students use the concept of membrane capacitance to calculate the number of ions that cross the membrane. They learn where these ions are located and why the bulk solutions conform to the principle of electroneutrality.

Apoptosis in C3H-10T1/2 cells: roles of intracellular pH, protein kinase C, and the Na+/H+ antiporter.

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whether intracellular pH could play a significant role in apoptotic initiation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na+/H+ antiporter, known regulators of intracellular pH, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H+ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na+/H+ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na+/H+ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na+/H+ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity.

Cytotoxicity of solid lipid nanoparticles as a function of the lipid matrix and the surfactant.

PURPOSE: Assessment of the in vitro cytotoxicity of solid lipid nanoparticles (SLNs) as a function of lipid matrix (Dynasan 114, Compritol ATO 888), and stabilizing surfactant (poloxamers, Tween 80, soya lecithin, and sodium dodecyl sulphate). Comparison with other colloidal carriers should determine their potential use in the clinic. METHODS: SLNs were produced by high pressure homogenisation. Cytotoxicity was assessed by measuring the viability of HL60 cells and human granulocytes after incubation with SLNs. Particle internalisation was quantified by chemiluminescence measurements. RESULTS: The nature of the lipid had no effect on viability; distinct differences were found for the surfactants. Binding to the SLN surface reduced markedly the cytotoxic effect of the surfactants, e.g., up to a factor of 65 for poloxamer 184. The permanent HL60 cell line-differentiated from cells with granulocyte characteristics by retinoic acid treatment-yielded results identical to freshly isolated human granulocytes. In general, the SLNs showed a lower cytotoxicity compared to polyalkylcyanoacrylate and polylactic/glycolic acid (PLA/ GA) nanoparticles. CONCLUSIONS: Because the results are identical when using human granulocytes, differentiated HL60 cells can be used as an easily accessible in vitro test system for i.v. injectable SLN formulations. The SLNs appear suitable as a drug carrier system for potential intravenous use due to their very low cytotoxicity in vitro.