Immunogenicity of an interferon-beta1a product.

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Aortic dissection: a review of the diagnosis and initial management.

Aortic dissection is a medical emergency which carries a high mortality, particularly if undiagnosed and untreated. The diagnosis may be overlooked because the 'classical' history, examination and imaging signs are present in only a minority of cases. After reflecting on the underlying pathophysiology, this review examines aspects of the history and examination which should alert Acute Physicians to a possible diagnosis of aortic dissection. The relative benefits of different imaging techniques are summarised, along with the prognosis and treatment for different types of aortic dissection. Finally a case of aortic dissection which went undiagnosed for a month is presented to illustrate some of the key teaching points raised in this article.

Plasmid-mediated VEGF gene transfer induces cardiomyogenesis and reduces myocardial infarct size in sheep.

We have recently reported that in pigs with chronic myocardial ischemia heart transfection with a plasmid encoding the 165 isoform of human vascular endothelial growth factor (pVEGF165) induces an increase in the mitotic index of adult cardiomyocytes and cardiomyocyte hyperplasia. On these bases we hypothesized that VEGF gene transfer could also modify the evolution of experimental myocardial infarct. In adult sheep pVEGF165 (3.8 mg, n=7) or empty plasmid (n=7) was injected intramyocardially 1 h after coronary artery ligation. After 15 days infarct area was 11.3+/-1.3% of the left ventricle in the VEGF group and 18.2+/-2.1% in the empty plasmid group (P<0.02). The mechanisms involved in infarct size reduction (assessed in additional sheep at 7 and 10 days after infarction) included an increase in early angiogenesis and arteriogenesis, a decrease in peri-infarct fibrosis, a decrease in myofibroblast proliferation, enhanced cardiomyoblast proliferation and mitosis of adult cardiomyocytes with occasional cytokinesis. Resting myocardial perfusion (99mTc-sestamibi SPECT) was higher in VEGF-treated group than in empty plasmid group 15 days after myocardial infarction. We conclude that plasmid-mediated VEGF gene transfer reduces myocardial infarct size by a combination of effects including neovascular proliferation, modification of fibrosis and cardiomyocyte regeneration.

Reversible acute myocardial injury following air bag deployment.

This case report is about a 62 year old woman who was involved in an accident while driving her car, during which the driver side air bag deployed. She experienced intense anterior chest pain that radiated to her left arm after the accident, but was otherwise well; there was no significant medical history. An electrocardiogram done one and half hours after admission revealed 1 mm ST segment elevation in leads V2 and V3 and troponin 1 level was raised. She underwent cardiac catheterisation but three months after the accident both ECG and echocardiographic studies were normal. It is suggested that she underwent cardiac contusion rather than a myocardial infarction.

Cardiac pacing systems and implantable cardiac defibrillators (ICDs): a radiological perspective of equipment, anatomy and complications.

Cardiac pacing is a proven and effective treatment in the management of many cardiac arrhythmias. Implantable cardiac defibrillators (ICDs) are beneficial for certain patient groups with a history of serious, recurrent ventricular dysrhythmias, with a high risk of sudden cardiac death. Pacemaker devices take many forms and are highly visible on the chest radiograph. The radiographic appearances of ICDs and pacemakers can be similar and are subject to similar complications. The anatomical approach to the implantation, the type of device used and anatomical variations will all affect the appearance of these devices on the chest film. Pacemaker complications identified radiographically include pneumothorax, lead malpositioning, lead displacement or fracture, fracture of outer conductor coil, loose connection between the lead and pacemaker connector block, lack of redundant loops in paediatric patients and excessive manipulation of the device by the patient (Twiddler's syndrome). This pictorial review highlights the role of chest radiography in the diagnosis of post-cardiac pacing and ICD insertion complications, as well as demonstrating the normal appearances of the most frequently implanted devices.

Entrance in mitosis of adult cardiomyocytes in ischemic pig hearts after plasmid-mediated rhVEGF165 gene transfer.

Replacement of the cell loss occurring after acute myocardial infarction has been proposed as a potential treatment to prevent heart remodeling and failure. On account that cardiomyocytes express VEGF receptors and that VEGF triggers mitogen-activated protein kinases, we investigated if VEGF gene transfer may induce cardiomyocyte replication. In a pig model of chronic myocardial ischemia achieved by Ameroid occlusion of the left circumflex coronary artery, we observed that direct intramyocardial injection of a plasmid encoding human VEGF(165) induced a several-fold increase in cardiomyocyte mitotic index and in the number of cardiomyocyte nuclei per unit volume as compared with pigs receiving plasmid devoid of gene. Despite images of conventional cytokinesis were not observed, the fact that caryokinesis is an obligatory step for cell division suggests that our finding may contribute to the issue of heart regeneration and may potentially widen the therapeutic spectrum of VEGF gene transfer.

Early experience with transmission of data from moving ambulances to improve the care of patients with myocardial infarction.

We conducted a feasibility study of a mobile unit capable of recording a 12-lead electrocardiogram (ECG) and transmitting it to a receiving hospital workstation. Two ambulances were equipped with the mobile unit and the ECGs recorded were compared with standard ECGs recorded on the hospital ward after admission. In six months, 62 patients participated in the study. The ambulance crew transmitted messages to the coronary care unit for 56 patients. Thirty-five patients were directed to the coronary care unit, four were directed to the emergency department as no beds were available and then transferred to the coronary care unit later, and the remaining 23 were directed to the emergency department. Comparative hospital and mobile ECGs were available for 31 patients. Although the mobile unit recorded smaller R- and S-wave deflections than on the standard hospital ECGs, the medical and nursing staff were able to differentiate between normal and abnormal tracings. The mobile unit may be useful to triage patients with chest pain before they reach hospital.

Immunologic and genetic markers in insulin-dependent diabetes mellitus (type 1) in an Argentine population.

The objective was to evaluate the prevalence and association of several markers (islet cell antibodies: ICA, insulin autoantibodies: IAA, glutamic acid decarboxylase antibodies: GADA and ICA512 antibodies: ICA512A) along with HLA DQB1 genotype in type 1 diabetes mellitus of recent onset, including siblings and individuals without any history of this disease, in an Argentine population. A total of 79 children with type 1 diabetes mellitus of recent onset were studied, as well as 79 control children, and 68 healthy siblings of type 1 diabetic cases. IAA, ICA, GADA, ICA512A and HLA DQB1 alleles were determined. Sensitivity was 67.1% for ICA, 36.7% for IAA, 74.6% for GADA and 63.4% for ICA512A. None of the control subjects was positive for the immunological markers. Combined sensitivity of ICA-IAA-GADA was 89.8%, similar to the ICA512A-GADA (87.3%) or ICA512A-GADA-IAA combination (91.1%). GADA correlated positively with ICA, but no such correlation was found between IAA, ICA512A and ICA. IAA correlated negatively and GADA positively with age. IAA was associated to DQB1*0201, whereas ICA and ICA512A associated to DQB1*0302. Among siblings, 3/68 (4.4%) were positive for IAA and a single case (1.5%) was positive for GADA and one for ICA512A. Our findings show that the combination of multiple tests increases the sensitivity for prediction, with the ICA512A-GADA combination proving highly sensitive and equivalent to other proposed combinations, such as ICA-IAA-GADA.

Immunologic and genetic markers in insulin-dependent diabetes mellitus (type 1) in an Argentine population.

The objective was to evaluate the prevalence and association of several markers (islet cell antibodies: ICA, insulin autoantibodies: IAA, glutamic acid decarboxylase antibodies: GADA and ICA512 antibodies: ICA512A) along with HLA DQB1 genotype in type 1 diabetes mellitus of recent onset, including siblings and individuals without any history of this disease, in an Argentine population. A total of 79 children with type 1 diabetes mellitus of recent onset were studied, as well as 79 control children, and 68 healthy siblings of type 1 diabetic cases. IAA, ICA, GADA, ICA512A and HLA DQB1 alleles were determined. Sensitivity was 67.1

for ICA, 36.7

for IAA, 74.6

for GADA and 63.4

for ICA512A. None of the control subjects was positive for the immunological markers. Combined sensitivity of ICA-IAA-GADA was 89.8

, similar to the ICA512A-GADA (87.3

) or ICA512A-GADA-IAA combination (91.1

). GADA correlated positively with ICA, but no such correlation was found between IAA, ICA512A and ICA. IAA correlated negatively and GADA positively with age. IAA was associated to DQB1*0201, whereas ICA and ICA512A associated to DQB1*0302. Among siblings, 3/68 (4.4

) were positive for IAA and a single case (1.5

) was positive for GADA and one for ICA512A. Our findings show that the combination of multiple tests increases the sensitivity for prediction, with the ICA512A-GADA combination proving highly sensitive and equivalent to other proposed combinations, such as ICA-IAA-GADA.

Long-term survival of VDD pacing.

UNLABELLED: All patients with VDD systems implanted at a tertiary pacing center were identified from a computer database and data collected on pacing indications, follow-up duration, rate response, reasons for programming changes, and implant P wave amplitudes. RESULTS: 366 implants were identified for which complete data were available for 335 leads implanted in 316 patients. The mean follow-up period was 24.1 months, and age at implant was 73.5 +/- 11.8 years. During follow-up, 19 patients died (6%) and 62 (19.6%) were followed elsewhere. Indications for pacing were complete heart block, 56.6%; intermittent AV block, 21.8%; postablation complete heart block, 5.4%; 2:1 AV block, 13%; and others, 3.2%. Two groups: no mode change (NMC, n = 280) and mode change (MC, n = 36) were identified. Reasons for reprogramming in the MC group were as follows: atrial sensing, 11; AF/atrial flutter, 18; chronotropic incompetence, 3; and others 4. Significantly more MC patients had rate response programmed ON (44.4% vs 22.1%, P < 0.05). No significant differences between the two groups were found in other variables, including male gender (55.5% vs 54.6%), length of follow-up (27.1 +/- 17.8 vs 23.8 +/- 20.6 months), age at last follow-up (72 +/- 12.3 vs 75.9 +/- 11.9 years), and P wave amplitude (1.7 +/- 0.9 vs 1.8 +/- 0.9 mV). CONCLUSION: Reprogramming of VDD systems is infrequent. When necessary, it is usually prompted by atrial arrhythmias or failure of atrial sensing. When adequate atrial chronotropy has been verified, VDD is an acceptable alternative to DDD pacing and survives well over the long term.

Highly-sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen.

Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.

Expression of properly folded human glutamate decarboxylase 65 as a fusion protein in Escherichia coli.

Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.

Evolution of atrial signals from a single lead VDD pacemaker. AddVent European Multicentre Study Group.

The atrial sensing capabilities of a new single pass lead VDD pacing system (Pacesetter AddVent) were assessed in a prospective multicenter study of 101 implants during the period July 1994 through March 1996. The pacing lead (Pacesetter AV Plus) has a unique quadripolar 4-in-line connector and uses a pair of ring electrodes with an interelectrode spacing of 12 mm for atrial sensing. The mean age of the patients (51 men) was 73 years (range 19-91). Seventy-five patients had complete heart block; the others had 2:1 AV block. Wide variations were found in signal amplitude: mean P wave amplitude, measured over four cycles in the supine position, was 2.4 +/- 1.9 mV at implant, dropping to 1.9 +/- 1.7 mV predischarge, and remaining constant at follow-up but with a narrower range. Holter monitoring was undertaken in 24 patients, with a total of 550 monitored hours. Mean AV synchrony was 98.2% +/- 4.6% (excluding premature ventricular contractions), with 20 patients (83%) showing > 99% AV synchrony, with atrial sensing at 0.1 mV where needed. No oversensing was observed in any patient. There was a low incidence of atrial fibrillation (2%) and sinus bradycardia (0%). The findings show that the range of atrial signals, although wide initially, converges over the first year and remains adequate for reliable AV synchronous pacing.

Detection of glutamic acid decarboxylase (GAD) autoantibodies by indirect immunofluorescence using CHO cells expressing recombinant human GAD65.

The reaction between human glutamic acid decarboxylase (GAD65) expressed in CHO cells and GAD antibodies was studied by indirect immunofluorescence (IIF). The monoclonal antibodies GAD1 and GAD6, which recognize conformational and continuous GAD epitopes respectively, yielded distinct staining patterns. Twelve of 26 sera from newly-diagnosed insulin-dependent diabetes mellitus (IDDM) patients displayed a variety of anti GAD specific IIF images encompassing the two extremes observed with the monoclonal antibodies. None of 21 normal sera tested positive in this assay. As a control, the sera were tested by a reference immunoprecipitation (IP) assay using in vitro produced, folded 35S-GAD65. Only one of the patient sera reacted by IP using heat- and detergent-denatured 35S-GAD65 indicating that most of the auto-antibodies recognized only a folded antigen. Eleven patient sera were both IIF and IP anti-GAD-positive. The IIF reactivity of these sera was blocked by soluble GAD from brain extracts. One serum was positive only by IIF, and its reactivity was not blocked by soluble GAD. Eight sera were positive only by IP. Our results established differences in anti GAD antibodies in terms of their capacity to recognize human GAD65 in the context of transformed CHO cells compared with conventional IP assays. These differences should be considered in future attempts to improve the available assays for the detection of IDDM autoantibodies.

Effects of atrial natriuretic factor on norepinephrine release evoked by angiotensins II and III in the rat adrenal medulla.

Atrial natriuretic factor (ANF) effects on neuronal norepinephrine (NE) release evoked by angiotensin II (ANG II) or angiotensin III (ANG III) were studied in the rat adrenal medulla. ANF 10 nM diminished the increase of NE release induced by ANG II (1 microM), ANG III (1 microM) or 100 mM KCl. When 10 nM ANF was added to the medium containing KCl plus ANG II or KCl plus ANG III, the reduction of 3H-NE output by ANF was greater than when the atrial factor was added to the medium containing only ANG II or ANG III. Since both ANG II and ANG III have a physiological role on catecholamine metabolism, these peptides could modulate the adrenal medulla functions. ANG II and ANG III enhance NE release and decrease NE uptake in the rat adrenal medulla. Present results show that ANF is a physiological antagonist of both ANG II and ANG III, in the process of NE secretion. The interaction between ANF and the renin-angiotensin system could contribute to the regulation of the adrenal medulla catecholamines pathway and sympathetic activity.

Angiotensin III modulates noradrenaline uptake and release in the rat hypothalamus.

1. Effects of angiotensin III (A III) on 3H-noradrenaline (3H-NA) total, neuronal and non-neuronal uptake, 3H intracellular distribution and release were studied in vitro in the rat hypothalamus. 2. A III (1 microM) decreased total, neuronal and non-neuronal 3H-NA uptake when hypothalamic slices were incubated with 3H-NA for 30 min. A III effects on neuronal and non-neuronal 3H-NA uptake were determined in the presence of 100 microM hydrocortisone or 10 microM cocaine hydrochloride, respectively. The effect of A III on total 3H-NA uptake was blocked by 10 microM Ile7 angiotensin III (Ile7 A III), an antagonist at A III receptors. In contrast, 100 nM A III had no effect on 3H-NA uptake. 3. The study of the 3H-NA uptake time course showed that 1 microM AIII decreased NA uptake at 1, 3, 7, 15 and 30 min incubation. 4. In hypothalamic slices preloaded with 3H-NA for 30 min, 1 microM AIII increased the 3H content in the granular pool and decreased it in the cytosolic pool. 5. Spontaneous 3H release was also modified by 1 microM A III when hypothalami were preloaded with 3H-NA for 30 min. A III increased the spontaneous output of 3H. This effect was receptor-mediated since the effect of A III on 3H release was antagonized by Ile7 A III. 6. The present results suggest that the effects of A III on NA neurotransmission, may be involved in the regulation of central angiotensin effects such as blood pressure control, hydrosaline balance and dipsogenesis, through modulation of central sympathetic activity.

Atrial natriuretic factor modifies noradrenaline release in a sodium-free medium.

1. The effects of atrial natriuretic factor (ANF) on 3H-noradrenaline (3H-NA) release evoked by a sodium-free medium (SFM) were studied. The experiments were carried out in rat hypothalamic slices incubated in vitro. 2. ANF (1, 10 and 100 nM) decreased NA release evoked by the omission of sodium in a concentration-dependent way. When calcium was omitted from a SFM, NA output was partially diminished. However, if ANF was added to the SFM/calcium free medium NA secretion showed no modifications. 3. Present results suggest that, in rat hypothalamus, NA release evoked by Na+ omission is divided into two fractions: one independent of and the other dependent on extracellular calcium. In addition, ANF modifies NA release evoked by SFM dependent on extracellular calcium.

Modulation of the rat adrenal medulla norepinephrine secretion in a sodium-free medium by atrial natriuretic factor.

The effects of atrial natriuretic factor (ANF) on [3H]norepinephrine ([3H]NE) release evoked by a sodium-free medium (SFM) were studied. Experiments were performed in rat adrenal medulla slices incubated in vitro. Results showed that [3H]NE release evoked by the omission of Na+ was decreased by 10 nM ANF. In addition, when the Ca2+ was omitted from the SFM, NE output was partially diminished. Nevertheless, if ANF was added to SFM/Ca(2+)-free medium (CFM), NE secretion showed no modifications compared with SFM/CFM. Present results raise the hypothesis that two mechanisms could be involved in NE output evoked by a SFM in the rat adrenal medulla: one independent of and the other dependent on the extracellular calcium. Moreover, ANF only diminished NE secretion evoked by SFM dependent on extracellular calcium and did not modify calcium-independent NE release.