Search PubMed⌕ Search

Biomedical subjects

G Szabo

Publications and source records attributed to G Szabo.

At least 19 recordsLinked to original sources
PubMed2002

Near field optical microscopy in aqueous solution: implementation and characterization of a vibrating probe.

Near field optical microscopy (NSOM) is one of the possible solutions to circumvent the diffraction limit, but the control of the optical probe in solution has been a technical challenge for practical applications. Most recently, it has been shown that the pipette used in the scanning ion conductance microscope can be modified to form a high resolution near field optical probe. When combined with a novel distance modulation mechanism, a robust near field microscope can be constructed for operation in aqueous solution. In this paper, we present technical details of this design and a further characterization of the NSOM system for imaging in solution. Fundamental limitations of this approach in comparison to other systems are also discussed. Based on the current technology, it is concluded that better than 50 nm resolution should be achievable with this technique for fluorescence, as well as fluorescence resonance energy transfer, imaging of biological specimens.

Microscopy↗
PubMed2001

Characterization of AC mode scanning ion-conductance microscopy.

A scanning ion-conductance microscope (SICM) with a vibrating probe has been recently developed (vSICM). In this system, the amplitude of the AC ionic current is detected by using a lock-in amplifier locked to the vibration frequency of the probe. Such a scheme allows for a better control of the tip position because the AC ionic current is more sensitive to the probe-surface distance than the DC ionic current used previously. In this paper, we demonstrate the utility of this technique to the imaging of topographically rough specimens and high-resolution imaging over selected small areas. We also show that it is possible to record the DC ionic current simultaneously during the scan, which can reveal additional information not apparent in the images obtained with the AC ionic current.

Electric Conductivity↗
PubMed2001

Vertical bias in dendritic trees of non-pyramidal neocortical neurons expressing GAD67-GFP in vitro.

The neocortical neuropil has a strong vertical (orthogonal to pia) orientation, constraining the intracortical flow of information and forming the basis for the functional parcellation of the cortex into semi-independent vertical columns or 'modules'. Apical dendrites of excitatory pyramidal neurons are a major component of this vertical neuropil, but the extent to which inhibitory, GABAergic neurons conform to this structural and functional design is less well documented. We used a gene gun to transfect organotypic slice cultures of mouse and rat neocortex with the enhanced green fluorescent protein (eGFP) gene driven by the promoter for glutamic acid decarboxylase 67 (GAD67), an enzyme expressed exclusively in GABAergic cells. Many GAD67-GFP expressing cells were highly fluorescent, and their dendritic morphologies and axonal patterns, revealed in minute detail, were characteristic of GABAergic neurons. We traced 150 GFP-expressing neurons from confocal image stacks, and estimated the degree of vertical bias in their dendritic trees using a novel computational metric. Over 70% of the neurons in our sample had dendritic trees with a highly significant vertical bias. We conclude that GABAergic neurons make an important contribution to the vertical neocortical neuropil, and are likely to integrate synaptic inputs from axons terminating within their own module.

Animals↗
PubMed2001

Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis.

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, DeltaAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to DeltaAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, DeltaN489 (missing amino acids 6 to 489) and DeltaN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while DeltaN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.

Adenylate Cyclase Toxin↗
PubMed2001

A 12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation.

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.

Animals↗
PubMed2001

Tacrolimus and cyclosporine A inhibit allostimulatory capacity and cytokine production of human myeloid dendritic cells.

Myeloid dendritic cells (DCs) are pivotal in the recognition of alloantigens and, therefore, in the induction of allograft rejection. Induction of alloreactive T cell proliferation by myeloid DCs depends on the maturation of DCs, the expression of costimulatory molecules, and the cytokine environment. This study investigated the effects of tacrolimus and cyclosporine A (CsA) on DC maturation and allostimulatory capacity. Myeloid DCs were propagated from normal blood monocytes with interleukin (IL) 4 and GM-CSF for 7 days in the presence or absence of tacrolimus (FK506; 10 nM) or CsA (1 microg/mL). Exposure of DCs during maturation to tacrolimus or CsA resulted in no significant change in the expression of DC phenotypic markers, including CD80, CD86, and HLA Class I and II antigens determined by flow cytometry. T cell proliferation in one-way, mixed-leukocyte reaction experiments revealed a decreased allostimulatory capacity of DCs that matured in the presence of tacrolimus or CsA compared with untreated controls (P<0.02). Production of inflammatory cytokines, tumor necrosis factor alpha (P<0.04) and IL-12 (P<0.04) in response to lipopolysaccharide (1 microg/mL) or staphylococcal enterotoxin B (1 microg/mL) induction was significantly reduced in DCs exposed to tacrolimus or CsA during maturation. In contrast, production of the immuninhibitory cytokine IL-10 was not decreased in tacrolimus- or CsA-treated DCs. These results suggest that tacrolimus and CsA inhibit the allostimulatory capacity of in vitro-generated myeloid DCs without significant effects on DC phenotypic maturation. Decreased production of IL-12 and tumor necrosis factor alpha, but not of IL-10, is likely to contribute to the impaired accessory-cell function of tacrolimus- and CsA-treated DCs. Thus, tacrolimus and CsA can inhibit recognition of alloantigens by decreasing the accessory-cell capacity of monocyte-derived myeloid DCs.

Cyclosporine↗
PubMed2001

Interferon alpha and alcohol augment nuclear regulatory factor-kappaB activation in HepG2 cells, and interferon alpha increases pro-inflammatory cytokine production.

BACKGROUND: The mechanisms for decreased therapeutic response to IFNalpha in chronic hepatitis C patients with alcohol are unknown. We investigated the hypothesis that IFNalpha and alcohol regulate cells both in the liver parenchyma and the immune system. METHODS: We used the hepatocellular carcinoma cells (HepG2) to determine if IFNalpha (500-10,000 U/ml) or ethanol (25-100 mM) modulates NF-kB activation alone or in combination with TNFalpha (0.1-20 microg/ml) as determined in electromobility gel shift assays. IkB levels were evaluated in the cytoplasmic extracts by western blot. Monocytes from normal donors were activated with LPS (1 microg/ml) in combination with IFNalpha or ethanol overnight and TNFalpha, IL-6, and IL-12 were measured in the supernatants. RESULTS: In HepG2 cells, both IFNalpha and acute alcohol treatment induced NF-kappaB activation and augmented TNFalpha-induced NF-kappaB binding. Pretreatment of HepG2 cells with IFNalpha resulted in the highest levels of NF-kappaB activation in response to TNFalpha or TNFalpha plus ethanol stimulation. Supershift experiments confirmed that the NF-kappaB dimer induced by TNFalpha and its combination with IFNalpha or ethanol contains RelA (p65) and involves rapid degradation of IkappaBalpha. Experiments using the proteasome inhibitor, MG132, revealed that augmentation of NF-kappaB by ethanol and IFNalpha is mediated via the proteasome pathway. We show that in normal monocytes, IFNalpha augments LPS-induced production of the inflammatory cytokines TNFalpha, IL-6, and IL-12 (p < 0.06) without further modulation by acute alcohol treatment. CONCLUSIONS: These results suggest that IFNalpha can increase HepG2 cell sensitivity to TNFalpha and ethanol-mediated activation. Augmentation of monocyte inflammatory cytokines, particularly of IL-12 production, by IFNalpha could be a key element of the antiviral response in chronic HCV. These results support the hypothesis that the therapeutic benefits of IFNalpha likely involve activation of both immune and parenchymal cells in the liver.

Carcinoma, Hepatocellular↗
PubMed2001

Reduced alloreactive T-cell activation after alcohol intake is due to impaired monocyte accessory cell function and correlates with elevated IL-10, IL-13, and decreased IFNgamma levels.

BACKGROUND: Immunosuppression associated with chronic alcohol use is characterized by reduced antigen-specific T-cell response and impaired delayed type hypersensitivity. Increasing evidence suggests in chronic alcohol consumption models that reduced antigen-specific T-cell proliferation is due to insufficient accessory cell function. Accessory cell function, a critical step in recognition of viral antigens, is reduced in chronic hepatitis C. The severity of hepatitis C is increased by alcohol consumption. Thus, we investigated the effects of alcohol consumption on accessory cell activity of monocytes in supporting alloreactive T-cell proliferation. METHODS: Alloreactive T-cell proliferation was evaluated in a one-way mixed lymphocyte reaction (MLR). Mononuclear cells were isolated by Ficoll density gradient and monocytes by adherence. Alcohol (0.8 g/kg body weight, an equivalent of approximately three drinks) was given to nonalcohol-consuming individuals and blood samples were collected before, 4 hr, or 18 hr after alcohol consumption. Alcohol in vitro was administered at concentrations of 25-100 mM. RESULTS: T-cell proliferation in MLR was significantly reduced in the presence of physiologically relevant concentrations of alcohol in vitro (25-100 mM ethanol) (p < 0.05). In vivo alcohol consumption also depressed proliferation in the MLR when stimulator cells were obtained 4 hr after alcohol consumption. MLR was not decreased, however, in the presence of alcohol-exposed responder cells and normal stimulator cells, suggesting that the accessory cell population and not T cells are affected by alcohol. Decreased accessory cell function was further evidenced by reduced superantigen-induced (SEB) but not mitogen-induced (PHA) T-cell proliferation in samples obtained 18 hr after alcohol intake (35% reduction). Reduced accessory cell function was not due to changes in surface expression of monocyte costimulatory molecules (HLA class I, HLA-DR, CD80, CD86, CD40). We found reduced IFNgamma, elevated IL-10, and unchanged IL-4 levels during T-cell proliferation in samples obtained 18 hr after alcohol consumption. Acute alcohol treatment resulted in increased IL-13 in the MLR. CONCLUSION: These data suggest that even on one occasion moderate alcohol intake can reduce allostimulatory T-cell activation via decreasing accessory cell function. Increased IL-10 and IL-13 plus the reduced IFNgamma production after acute alcohol use are likely to contribute to both the reduced T-cell proliferation and monocyte accessory cell function. These accessory cell mediated defects in T-cell activation may result in impaired antiviral and antitumor immunity after moderate acute alcohol use.

Adult↗
PubMed2000

Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers.

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood. Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial. We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements. In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels. These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane. The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels. The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA. Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers.

Bacterial Outer Membrane Proteins↗
PubMed2000

Acceptance of an ABO-incompatible mismatched (AB(+) to O(+)) liver allograft with the use of daclizumab and mycophenolate mofetil.

Liver allograft survival rates of 50% to 60% are reported in blood group A, group B, group O (ABO)-incompatible mismatched grafts even when aggressive immunosuppressive protocols, including plasmapheresis, OKT(3), cyclophosphamide, cyclosporine, prostaglandin E(1), and steroids, are used. A 59-year-old woman, blood type O(+), required emergency retransplantation posttransplantation day 2 because of primary nonfunction of the liver allograft. A blood type AB(+) allograft was used. Induction immunosuppressive therapy included tacrolimus, mycophenolate mofetil, OKT(3) (muromonab-CD(3)), steroids, and prostaglandin E(1). In addition, plasmapheresis was performed daily for 9 days. OKT(3) and prostaglandin E(1) were also discontinued postoperative day 9. Biopsy-proven acute cellular rejection was diagnosed postoperative day 12 and was treated with double-dose OKT(3) (10 mg) for another 6 days. On the day OKT(3) was discontinued, daclizumab, 60 mg, was administered intravenously. This dose was repeated every 2 weeks for a total of 5 doses. At 1-year follow-up, the patient is doing very well with normal liver function. We are unaware of previous reports of the use of daclizumab and mycophenolate mofetil as part of an immunosuppressive protocol aimed to induce acceptance of ABO-incompatible mismatched liver allografts. Based on our experience with this case, it seems that mycophenolate mofetil is an adequate replacement for cyclophosphamide. We also believe daclizumab provided adequate protection at a critical time. Further experience with both these drugs is required to establish their role in ABO-incompatible mismatched liver allografts.

Antibodies, Monoclonal↗
PubMed2000

Vascular tracheobronchial compression syndromes-- experience in surgical treatment and literature review.

Between January 1988 and December 1997 a total of 22 patients (age: 8 days-46 years) were operated for vascular airway compression syndromes with respiratory insufficiency. Vascular anomalies in tracheal compression were double aortic arch in 7 patients, (2 previously operated elsewhere), right aortic arch + left ligamentum arteriosum in 1, and pulmonary artery sling in 3. Three of these patients had secondary long-segment tracheomalacia. Compression of trachea and a main bronchus existed in 2 patients with right aortic arch + left ligamentum. Isolated main bronchus obstruction was present in 9 patients (abnormal insertion of ligamentum arteriosum in 1, status post (s.p.) previous operation for PDA in 4, s. p. surgery for coarctation in 1, right aortic arch + left ligamentum arteriosum in 2, and right lung aplasia + left ligamentum in 1). 3 of these cases had secondary long-segment bronchomalacia. All patients had a complex respiratory anamnesis [long-term intubation in 7, s.p. tracheostomy in 2 (over 3 months - 3 years), and progressive respiratory insufficiency in 13). In tracheal compression, surgical correction included transsection of the underlying ring or sling components (with additional anterior aortic arch translocation in 5 patients resection-reimplantation of left pulmonary artery in 3, segmental tracheal resection in 1, and external tracheal suspension in 2). In the 2 cases with compression of the trachea and a main bronchus, aortic "extension" by a prosthetic tube was necessary. In isolated main bronchus obstruction, surgical decompression basically consisted of transsection of the ligamentum arteriosum or resection of its scarry remnant forming the "corner point" of a compression between aorta and pulmonary artery. In 3 patients with secondary long-segment malacia, additional external bronchus suspension was performed. Effective decompression and re-expansion of the airway segment concerned was achieved, and was demonstrated by intraoperative endoscopy in all patients. There were 3 postoperative deaths (sepsis 2; massive, irreversible edema of the tracheal mucosa 1). Of the 19 surviving patients 16 could be extubated between the 1st and 17th (mean = 7.5) postoperative day. In 1 case the preoperative long-term tracheostomy had to be left in place for inoperable additional laryngeal stricture. 2 patients had to be reoperated (segmental cervical tracheal resection after 5 months for primary long-term intubation-related subglottic stenosis in 1, esophageal decompression for residual dysphagia after 57 months related to a traction phenomenon at the right descending aorta in the other), both with gratifying results. In all other patients clinical, endoscopic, and radiographic examinations (follow-up = 2 months - 6 years) demonstrate good results.

Adolescent↗
PubMed2000

Spatial evolutionary prisoner's dilemma game with three strategies and external constraints

The emergency of mutual cooperation is studied in a spatially extended evolutionary prisoner's dilemma game in which the players are located on the sites of cubic lattices for dimensions d=1, 2, and 3. Each player can choose one of the three following strategies: cooperation (C), defection (D) or "tit for tat" (T). During the evolutionary process the randomly chosen players adopt one of their neighboring strategies if the chosen neighbor has a higher payoff. Moreover, an external constraint imposes that the players always cooperate with probability p. The stationary state phase diagram is computed by both using generalized mean-field approximations and Monte Carlo simulations. Nonequilibrium second-order phase transitions associated with the extinction of one of the possible strategies are found and the corresponding critical exponents belong to the directed percolation universality class. It is shown that externally forcing the collaboration does not always produce the desired result.

Journal Article↗
PubMed2000

Branching annihilating random walk on random regular graphs

The branching annihilating random walk is studied on a random graph whose sites have a uniform number of neighbors (z). The Monte Carlo simulations in agreement with the generalized mean-field analysis indicate that the concentration decreases linearly with the branching rate for z>/=4, while the coefficient of the linear term becomes zero if z=3. These properties are described by a modified mean-field theory taking explicitly into consideration the probability of mutual annihilation of the parent and its offspring particles using the returning features of a single walker on the same graph.

Journal Article↗
PubMed2000

Management of recurrent hepatitis C after liver transplantation: a concise review.

Recurrent hepatitis C infection and subsequent graft failure are increasingly recognized problems after orthotopic liver transplantation. Although many prospective therapeutic, controlled trials in primary hepatitis C disease have been reported, large-scale studies are yet to be performed in patients with posttransplant recurrent hepatitis C after liver transplantation. In this review, we summarize the current literature on the therapeutic approaches for recurrent hepatitis C and discuss the results of published studies on therapy with ribavirin or interferon (IFN) alone and on combination therapy with IFN plus ribavirin. Further, we discuss results of prophylactic approaches to the problem of recurrent hepatitis C after transplant. Finally, we discuss additional aspects of anti-hepatitis C virus therapy after liver transplantation.

Antiviral Agents↗
PubMed2000

Variable absorption of carbidopa affects both peripheral and central levodopa metabolism.

Carbidopa (CD), a competitive inhibitor of aromatic l-amino acid decarboxylase that does not cross the blood-brain barrier, is routinely administered with levodopa (LD) to patients with Parkinson disease (PD) to reduce the peripheral decarboxylation of LD to dopamine. Using a stable isotope-labeled form of LD, the authors examined in 9 PD patients the effects of variable CD absorption on peripheral and central LD metabolism. Subjects were administered orally 50 mg of CD followed in 1 hour by a slow bolus intravenous infusion of 150 mg stable isotope-labeled LD (ring 1',2',3',4',5',6'-13C). Eight patients underwent a lumbar puncture 6 hours following the infusion. Blood and cerebrospinal fluid (CSF) samples were analyzed for labeled and unlabeled metabolites using a combination of high-performance liquid chromatography and mass spectrometry. When patients were divided into "slow" and "rapid" CD absorption groups, significantly greater peripheral LD decarboxylation (as measured by area under the curve [AUC]-labeled serum HVA) was noted in the poor absorbers (p = 0.05, Mann-Whitney U test). Elimination half-lives for serum LD did not differ between groups, suggesting a further capacity for decarboxylation inhibition in the "rapid" absorbers. A significant correlation between AUC serum CD and percent-labeled HVA in CSF was found for all patients (R = 0.786, p = 0.02). "Rapid" as compared to "slow" CD absorbers had significantly more percent-labeled CSF HVA (60 vs. 49, p = 0.02, Mann-Whitney U test), indicating greater central-labeled DA production in the better CD absorbers. The data suggest that peripheral aromatic l-amino acid decarboxylase activity is not saturated at CD doses used in current practice. The authors believe that future studies to better examine a dose dependence of CD on peripheral LD decarboxylation and LD brain uptake are warranted.

Absorption↗
PubMed2000

A lumped parameter model of left ventricular filling-pressure waveforms.

Assessment of left-ventricular function and quantification of valve stenosis are important in clinical practice as well as in physiological research. This paper describes a mathematical model of diastolic ventricular filling and flow across the mitral valve. The model primarily consists of a system of three first-order, non-linear ordinary differential equations. The equations are solved in MATLAB using the ode45 command. The model generates pressure versus time waveforms for the atrium and ventricle, and flow versus time waveforms for mitral valve flow. Comparisons between model outputs and in-vivo data collected in two porcine experiments show excellent agreement.

Diastole↗