Development of cellulose-DNA immunoadsorbent

The aim of this study was to prepare a DNA immunoadsorbent for the specific, extracorporeal removal of anti-DNA antibodies from the blood of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two kinds of cellulose beads were applied as a carrier. Calf thymus DNA was covalently coupled to the carrier using the epichlorohydrin method. Efforts were focused on optimization of conditions for activation and coupling, trying to couple as much DNA as possible to a certain amount of carrier. It was found that the activation level increased with the increase of NaOH concentration and the amount of epichlorohydrin used. The mole of epichlorohydrin must be in excess of that of NaOH because excess NaOH could react further with the epoxy groups in the beads resulting in a decrease of activation level. High activation level could be obtained in a medium of 3.0 M NaOH. The DNA coupling was found to be mainly temperature and pH dependent. Using 0.1 M Tris-HCl buffer, pH 8 at a temperature of 50-90 degrees C, more than 3 mg of DNA could be coupled to 1 ml of wet beads. Prolonging the coupling reaction under 50 degrees C to 72 h resulted in the same coupling capacity as that obtained under 90 degrees C. To evaluate the adsorption ability for anti-DNA of this immunoadsorbent, batch and circulation tests were applied using SLE patient plasma. The immunoadsorbents showed excellent adsorption capacity, especially the cellulose with smaller size (200-300 microm). The incubation of 20 ml of patient's plasma with 1 ml of adsorbent resulted in an 80% decline in the anti-DNA antibody level. In the circulation tests, 30 ml of plasma was circulated through a column containing 3 ml of adsorbent. The maximum decline in anti-DNA level, 80%, was obtained after 60 min. Such high adsorption capacity and high adsorption rate suggest this immunoadsorbent may be used for treatment. For comparison, 1,4-butanediol diglycidyl ether activation method and other DNA sources were tested with the same protocol.     

Fibrinogen predicts restenosis after endovascular treatment of the iliac arteries

OBJECTIVE: Fibrinogen is an acute phase protein as well as a component of the coagulation cascade. Vascular inflammation and disturbed coagulation are suggested to cause restenosis after percutaneous transluminal angioplasty (PTA). We investigated the prognostic impact of fibrinogen on restenosis after endovascular treatment of iliac artery occlusive disease. METHODS: In a prospective cohort study 137 consecutive patients after iliac artery PTA (n = 74) and PTA plus selective stent implantation (n = 63) were included, 109 patients after lower limb angiography served as a control group. Patients were followed for 6 months with oscillography, ankle brachial index and duplex sonography for occurrence of restenosis. Fibrinogen and serum amyloid A (SAA), as a control parameter of inflammation, were obtained at baseline, 8, 24 and 48 h postintervention. RESULTS: PTA (adjusted OR 3.1, p = 0.05) and stenting (adjusted OR 13.3, p = 0.001) were independently associated with a higher postintervention increase of fibrinogen compared to angiography. Restenosis was found in 29 patients (21%). Patients with pre-intervention fibrinogen values in the third quartile (411-463 mg/dl) had a 6.2-fold increased adjusted risk for restenosis (p = 0.03), patients in the fourth quartile (> 463 mg/dl) had a 8.9-fold increased adjusted risk (p = 0.007). Pre-intervention SAA values were also significantly associated with restenosis (p < 0.0001). Postintervention fibrinogen and SAA levels showed no association with outcome. CONCLUSION: Balloon angioplasty and stenting of the iliac arteries cause an elevation of postintervention fibrinogen levels independently of angiographic factors. A higher pre-procedure fibrinogen level, presumably a marker of inflammatory activity, indicates a higher risk for restenosis.     

Combined effects of eptifibatide and anticoagulants: differences between LMWH and UH or rH in thrombin generation inhibition but not in platelet aggregation inhibition

Aim of our study was to investigate effects of eptifibatide and anticoagulants on platelet aggregation and thrombin generation under low and high coagulant challenge in tissue factor-activated platelet rich plasma using a model allowing simultaneous determination of the time course of platelet aggregation and thrombin generation. Eptifibatide exerted a dose-dependent anti-aggregating effect under both high and significantly stronger under low coagulant challenge. Combination of eptifibatide and anticoagulants resulted in significant additive prolongation of the lag phase until the onset of platelet aggregation, more pronounced under low coagulant challenge. Under high, but not under low coagulant challenge combination of eptifibatide and anticoagulants had a significant synergistic inhibitory effect on platelet aggregation. Under low coagulant challenge combination of eptifibatide with LMWH, but not with UH, or rH, resulted in significantly reduced thrombin potential, F 1+2 generation, and FXa formation compared to measurements in the absence of eptifibatide. We demonstrate a synergistic effect of eptifibatide and anticoagulants on platelet aggregation inhibition and an additional inhibitory effect of LMWH and eptifibatide on thrombin generation. Our results support the notion that combination of eptifibatide and anticoagulants might be beneficial in atherosclerotic disease to palliate the thrombogenic potency of ruptured atherosclerotic plaques.     

Transfer of tissue factor from platelets to monocytes: role of platelet-derived microvesicles and CD62P

Tissue factor (TF) is the most important initiator of intravascular coagulation. Platelets contribute to TF exposure on monocytes, but the mechanism is not completely understood. Here we examined the possibility that platelets may release TF that can be transferred to monocytes by platelet-derived microvesicles. When human citrated platelet-rich plasma was incubated with collagen there was an increase in the plasma levels of TF and CD62P. Incubation of plasma obtained from collagen-stimulated PRP with a sediment of red and white blood cells resulted in an increase in the number of monocytes that express TF, CD62P and the platelet-specific antigen CD42a on their surface. This transfer of platelet-derived antigens to monocytes was reduced when CD62P was blocked by a specific antibody or when platelet-derived microvesicles were removed from the plasma either by high speed centrifugation (17,500 x g for 30 min) or by filtration (pore size 0.2 microm). The data indicate that platelet-derived microvesicles that are released from collagen-stimulated platelets may carry TF, CD62P and CD42a and may transfer these antigens to the surface of monocytes. The interaction of platelet-derived microvesicles with monocytes and the transfer of TF to monocytes strongly depend on CD62P.     

Alzheimer's second patient: Johann F. and his family

Alois Alzheimer evaluated five cases of Alzheimer's disease in the early 20th century. We focused on the family of "Johann F.," Alzheimer's second patient, who died in October 1910 at age 57 years, and whose brain pathology is typical of a subgroup of Alzheimer's disease, the so-called "plaque-only type." It was perhaps Emil Kraepelin's personal knowledge of this patient and the histological data of the other four cases that influenced Kraepelin to coin the term Alzheimer's disease. The church archive in Passau has a genealogical database drawn from sacramental registers dating from approximately 1580 to 1900. The genealogical data of the "Johann F." family, which comes from villages in Lower Bavaria, extends as far back as 1670. We found documentation starting around 1830 about cause of death in the church records, which shows a familial predisposition to dementia. Affected family members include the mother, maternal grandfather, maternal great-aunt, maternal great-grandfather as well as three of Johann F.'s eight siblings. The offspring (children and grandchildren) of these affected siblings also were affected by mental illness. We conclude that "Johann F." represents the index case of a family with a predisposition to presenile dementia with variable age of onset (30s to 60s).     

Gliomatosis cerebri: molecular pathology and clinical course

Gliomatosis cerebri is a rare, diffusely growing neuroepithelial tumor characterized by extensive brain infiltration involving more than two cerebral lobes. Among 13 patients with gliomatosis cerebri (median age, 46 years), biopsies showed features of diffuse astrocytoma (n = 4), oligoastrocytoma (n = 1), anaplastic astrocytoma (n = 5), anaplastic oligoastrocytoma (n = 1), or glioblastoma (n = 2). Molecular genetic investigation showed TP53 mutations in three of seven tumors and both PTEN mutation and epidermal growth factor receptor overexpression in one tumor. Amplification of CDK4 or MDM2 or homozygous deletion of CDKN2A was not detected. Three of 10 patients receiving radiotherapy showed a partial response (one patient) or had stable disease (two patients) lasting for more than 1 year. Four of six patients treated with procarbazine, carmustine, vincristine chemotherapy demonstrated partial remission (one patient), minor response (two patients), or stable disease (one patient). Median survival time from diagnosis was 14 months (range, 4-91+ months). Infratentorial involvement was associated with shorter survival. We conclude that (1) the molecular genetic alterations in gliomatosis cerebri resemble those in diffuse astrocytomas; (2) the prognosis of gliomatosis cerebri is variable but for at least 50% of patients as poor as for glioblastoma; and (3) some patients respond to radiotherapy and/or procarbazine, carmustine, vincristine chemotherapy.     

Intracellular chloride modulates A-type potassium currents in astrocytes

Application of the GABA(A) receptor agonist muscimol to astrocytes in situ or in vitro results in a receptor-mediated Cl(-) current with a concomitant block of outward K(+) currents. The effect on K(+) current is largely selective for the inactivating A-type current. Parallel experiments with various Cl(-) pipette concentrations show a significant reduction in A-type current under low Cl(-) conditions with minimal effect on delayed current. In addition, lower Cl(-) conditions caused a depolarizing shift of steady-state inactivation (V(1/2), -68 to -57 mV) and activation (V(1/2), -5.8 to 34 mV) kinetics of A-type current only. Cl(-) had no effect on the time course of inactivation or reactivation kinetics, suggesting the Cl(-)-mediated effect is largely on activation kinetics, indirectly affecting steady-state inactivation. Muscimol application to astrocytes under perforated patch control (gramicidin) displayed a similar block of A-type current to that of conventional whole cell patch at 40 or 20 mM pipette Cl(-) concentrations. With barium application under perforated patch conditions, the study of muscimol-mediated Cl(-) current in isolation of the effect on K(+) currents was possible. This allowed estimation of intracellular Cl(-) concentration from receptor current reversal information. The average intracellular Cl(-) concentration was found to be 29 +/- 3.2 mM. The effect on activation kinetics and lack of effect on time course of inactivation or reactivation suggest that intracellular anion concentrations have an effect on the K(+) channel voltage sensor region. Cl(-) may modulate K(+) currents by altering membrane field potentials surrounding K(+) channel proteins.     

Cerebellum and speech perception: a functional magnetic resonance imaging study

A variety of data indicate that the cerebellum participates in perceptual tasks requiring the precise representation of temporal information. Access to the word form of a lexical item requires, among other functions, the processing of durational parameters of verbal utterances. Therefore, cerebellar dysfunctions must be expected to impair word recognition. In order to specify the topography of the assumed cerebellar speech perception mechanism, a functional magnetic resonance imaging study was performed using the German lexical items "Boden" ([bodn], Engl. "floor") and "Boten" ([botn], "messengers") as test materials. The contrast in sound structure of these two lexical items can be signaled either by the length of the wordmedial pause (closure time, CLT; an exclusively temporal measure) or by the aspiration noise of wordmedial "d" or "t" (voice onset time, VOT; an intrasegmental cue). A previous study found bilateral cerebellar disorders to compromise word recognition based on CLT whereas the encoding of VOT remained unimpaired. In the present study, two series of "Boden - Boten" utterances were resynthesized, systematically varying either in CLT or VOT. Subjects had to identify both words "Boden" and "Boten" by analysis of either the durational parameter CLT or the VOT aspiration segment. In a subtraction design, CLT categorization as compared to VOT identification (CLT - VOT) yielded a significant hemodynamic response of the right cerebellar hemisphere (neocerebellum Crus I) and the frontal lobe (anterior to Broca's area). The reversed contrast ( VOT - CLT) resulted in a single activation cluster located at the level of the supratemporal plane of the dominant hemisphere. These findings provide first evidence for a distinct contribution of the right cerebellar hemisphere to speech perception in terms of encoding of durational parameters of verbal utterances. Verbal working memory tasks, lexical response selection, and auditory imagery of word strings have been reported to elicit activation clusters of a similar location. Conceivably, representation of the temporal structure of speech sound sequences represents the common denominator of cerebellar participation in cognitive tasks acting on a phonetic code.