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Biomedical subjects

M A Schmidt

Publications and source records attributed to M A Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed2000

A stable and highly sensitive 3,3',5,5'-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays.

3,3',5,5'-Tetramethylbenzidine (TMB) is a widely used chromogen for horseradish peroxidase-based detection systems because it yields reaction products with high absorption coefficients and lacks carcinogenicity. Unfortunately, TMB is labile and poorly soluble in aqueous buffers and such solutions must be freshly prepared before each experiment. Moreover, substrate depletion can occur under assay conditions. To overcome these problems we have developed a two-component TMB substrate system which has a lower detection limit and is more sensitive than many commercially available TMB reagents when compared in microtiter plate enzyme-linked immunosorbent assays. Both components of the substrate system are stable for at least 1 year at 4 degrees C and the usual discoloration of TMB stock solutions is prevented by the addition of a stabilizer that decomposes upon mixing of the two components.

Animals↗
PubMed2000

Bacterial interplay at intestinal mucosal surfaces: implications for vaccine development.

The discovery of 'molecular syringes' in several important gastrointestinal pathogens including Escherichia coli, Salmonella, Shigella and Yersinia, together with a better understanding of M cells and the mucosal immune system, has advanced our appreciation of multistage microorganism-host cell interactions. Recent studies suggest that these molecular strategies could be adapted for the development of modular mucosal vaccines.

Bacterial Proteins↗
PubMed2000

Characterization of M cell development during indomethacin-induced ileitis in rats.

BACKGROUND: M cells play an important role in the intestinal immune system as they have a high capacity for transcytosis of a wide range of microorganisms and macromolecules. However, little is known about the role of M cells during intestinal inflammation. AIM: We studied M cell development during indomethacin-induced intestinal inflammation in rats. METHODS: Ileitis in rats was induced by two subcutaneous injections with indomethacin (7.5 mg/kg) given 24 h apart. Rats were sacrificed after 14 days and tissue was analysed by fluorescence microscopy and electron microscopy. M cells could be visualized by using the FITC-labelled mAb anti-cytokeratin (CK)-8 (clone 4.1.18), which was recently identified as specific M cell marker in rats. The number of cytokeratin-8 positive M cells was related to the surface of the follicle associated epithelium. For morphological studies, we used both transmission electron microscopy (T.E.M.) and scanning electron microscopy (S.E.M.). RESULTS: In non-inflamed ileum M cells were scarce. Only 4% of the follicle associated epithelium were M cells, whereas an increase of M cells up to 11% was found in inflamed follicle associated epithelium (P < 0.001). The rate of M cell induction depended on the macroscopic degree of inflammation. T.E.M./S.E.M. studies showed that in inflamed tissue most M cells underwent apoptosis with typical morphological signs. In contrast to apoptotic M cells, the neighbouring enterocytes usually appeared intact. The number of mononuclear cells below the follicle associated epithelium was significantly increased. S.E.M. studies revealed that during induced ileitis mononuclear cells migrated from the lamina propria into the gut lumen by passing through apoptotic M cells. CONCLUSIONS: During indomethacin-induced ileitis in rats the increase in M cell number in association with apoptosis of M cells may alter the intestinal barrier function. These observations may play a pivotal role in the pathogenesis of chronic intestinal inflammation, e.g. in inflammatory bowel disease.

Animals↗
PubMed2000

Evaluation of patients undergoing lung volume reduction surgery: ancillary information available from computed tomography.

AIM: A number of imaging techniques have been used for the pre-operative assessment of patients for lung volume reduction surgery (LVRS). We evaluated whether data currently acquired from perfusion scintigrams and cine MR of the diaphragm are obtainable from high resolution CT (HRCT) of the thorax. MATERIALS AND METHODS: Thirty patients taking part in a randomized controlled trial of LVRS against maximal medical therapy were evaluated. HRCT examinations (n= 30) were scored for (i) the extent and distribution of emphysema; (ii) the extent of normal pulmonary vasculature; and (iii) diaphragmatic contour, apparent defects and herniation. On scintigraphy, (n= 28), perfusion of the lower thirds of both lungs, as a proportion of total lung perfusion (LZ/T(PERF)), was expressed as a percentage of predicted values (derived from 10 normal control subjects). On cine MR (n= 25) hemidiaphragmatic excursion and coordination were recorded. RESULTS: Extensive emphysema was present on HRCT (60% +/- 13.2%). There was strong correlation between the extent of normal pulmonary vasculature on HRCT and on perfusion scanning (r(s)= 0.85, P< 0.00005). Hemidiaphragmatic incoordination on MR was weakly associated with hemidiaphragmatic eventration on HRCT (P= 0.04). CONCLUSION: The strong correlation between lung perfusion assessed by HRCT and lung perfusion on scintigraphy suggests that perfusion scintigraphy is superfluous in the pre-operative evaluation of patients with emphysema for LVRS.

Adult↗
PubMed2000

Role of M cells in intestinal barrier function.

M cells are known as specialized epithelial cells of the follicle-associated epithelium of the gastrointestinal tract. As M cells have a high capacity for transcytosis of a wide range of microorganisms and macromolecules, they are believed to act as an antigen sampling system. The primary physiological role of M cells seems to be the rapid uptake and presentation of particular antigens and microorganisms to the immune cells of the lymphoid follicle to induce an effective immune response. In contrast to absorptive enterocytes, M cells do not exert direct defense mechanisms to antigens and pathogens in the gut lumen. Therefore, they provide functional openings of the epithelial barrier. Although M cells represent a weak point of the epithelial barrier, even under noninflamed conditions, there seems to be a balance between antigen uptake and immunological response. The low number of M cells in the gastrointestinal tract and the direct contact to immune cells in the lamina propria usually prevent the occurrence of mucosal inflammation. During chronic intestinal inflammation we observe an increase of M cell number and apoptosis selectively in M cells. M cell damage seems to be responsible for the increase of the uptake of microorganisms that is observed during intestinal inflammation. Under inflammatory conditions in the intestine, the maintenance of the epithelial barrier is broken and M cells seem to play a major role during this process.

Animals↗
PubMed2000

Correlations between antibody immune responses at different mucosal effector sites are controlled by antigen type and dosage.

Monitoring specific secretory immunoglobulin A (IgA) responses in the intestines after mucosal immunization or infection is impeded by the fact that sampling of small intestinal secretions requires invasive methods not feasible for routine diagnostics. Since IgA plasma cells generated after intragastric immunization are known to populate remote mucosal sites as well, secretory IgA responses at other mucosal surfaces may correlate to those in the intestines and could serve as proxy measures for IgA secretion in the gut. To evaluate the practicability of this approach, mice were immunized intragastrically with 0.2, 2, and 20 mg of ovalbumin plus 10 microg of cholera toxin, and the antigen-specific local secretory IgA responses in duodenal, ileal, jejunal, rectal, and vaginal secretions, saliva, urine, and feces, as well as serum IgG and IgA responses were analyzed by enzyme-linked immunosorbent assay. Correlation analysis revealed significant relationships between serum IgG and IgA, urinary IgA, salivary IgA, and secretory IgA in duodenal, jejunal, ileal, and rectal secretions for the 0.2-mg but not for the 20-mg ovalbumin dose. Fecal samples were poor predictors for intestinal antiovalbumin IgA responses, and no correlations could be established for cholera toxin, neither between local anti-cholera toxin levels nor to the antiovalbumin responses. Thus, specific IgA in serum, saliva, or urine can serve as a predictor of the release of specific IgA at intestinal surfaces after intragastric immunization, but the lack of correlations for high ovalbumin doses and for cholera toxin indicates a strong dependency on antigen type and dosage for these relationships.

Animals↗
PubMed1999

Real-time three-dimensional echocardiography for measurement of left ventricular volumes.

Left ventricular (LV) volumes are important prognostic indexes in patients with heart disease. Although several methods can evaluate LV volumes, most have important intrinsic limitations. Real-time 3-dimensional echocardiography (RT3D echo) is a novel technique capable of instantaneous acquisition of volumetric images. The purpose of this study was to validate LV volume calculations with RT3D echo and to determine their usefulness in cardiac patients. To this end, 4 normal subjects and 21 cardiac patients underwent magnetic resonance imaging (MRI) and RT3D echo on the same day. A strong correlation was found between LV volumes calculated with MRI and with RT3D echo (r = 0.91; y = 20.1 + 0.71x; SEE 28 ml). LV volumes obtained with MRI were greater than those obtained with RT3D echo (126 +/- 83 vs 110 +/- 65 ml; p = 0.002), probably due to the fact that heart rate during MRI acquisition was lower than that during RT3D echo examination (62 +/- 11 vs 79 +/- 16 beats/min; p = 0.0001). Analysis of intra- and interobserver variability showed strong indexes of agreement in the measurement of LV volumes with RT3D echo. Thus, LV volume measurements with RT3D echo are accurate and reproducible. This technique expands the use of ultrasound for the noninvasive evaluation of cardiac patients and provides a new tool for the investigational study of cardiovascular disease.

Adult↗
PubMed1999

Comparison of four 64Cu-labeled somatostatin analogues in vitro and in a tumor-bearing rat model: evaluation of new derivatives for positron emission tomography imaging and targeted radiotherapy.

Previous studies have shown that modification of the somatostatin analogue octreotide (OC), by substitution of tyrosine for phenylalanine at position 3 and of a C-terminal carboxylic acid for an alcohol, to give Tyr3-octreotate (Y3-TATE) improved uptake of the peptide in somatostatin receptor-positive tissues. To determine which substitution best accounts for increased target tissue uptake, the peptides containing single modifications, Tyr3-octreotide (Y3-OC) and octreotate (TATE), were synthesized. These peptides were conjugated to the macrocyclic chelating agent 1,4,8, 11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA) and radiolabeled with 64Cu(II). The in vitro receptor binding, in vitro tumor cell uptake, and in vivo distribution properties of 64Cu-labeled TETA-Y3-OC and TETA-TATE were compared to those of [64Cu]TETA-OC and [64Cu]TETA-Y3-TATE. Cu-TETA-TATE (IC50 = 0.297 +/- 0.0055 nM) and Cu-TETA-Y3-TATE (IC50 = 0.308 +/- 0.0375 nM) displayed significantly higher binding affinity to somatostatin receptors on CA20948 rat pancreatic tumor membranes than Cu-TETA-Y3-OC (IC50 = 0.397 +/- 0.0206 nM) and Cu-TETA-OC (IC50 = 0. 498 +/- 0.039 nM). Similarly, the uptakes of [64Cu]TETA-Y3-TATE (60. 75 +/- 1.21%) and [64Cu]TETA-TATE (55.62 +/- 0.16%) into AR42J rat pancreatic tumor cells over a 2-h time period were higher than those of [64Cu]TETA-Y3-OC (47.20 +/- 1.20%) and [64Cu]TETA-OC (34.07 +/- 2. 24%). The in vitro results suggest that the C-terminal carboxylate may contribute more to enhanced receptor binding and tumor cell uptake than the substitution at the 3-position. Biodistributions in CA20948 tumor-bearing rats showed receptor-mediated uptake of the 64Cu-labeled peptides in somatostatin-rich tissues, including the pituitary, adrenals, pancreas, and tumor. The structure-activity relationships of the four 64Cu-labeled peptides did not show consistent trends in all target tissues, but [64Cu]TETA-Y3-TATE exhibited tumor uptake 1.75-3.5 times higher than the other derivatives at 4 h postinjection. The greater tumor retention of [64Cu]TETA-Y3-TATE justifies the selection of this agent for future PET imaging and targeted radiotherapy studies.

Adrenal Glands↗
PubMed1999

In vitro and in vivo evaluation of 64Cu-TETA-Tyr3-octreotate. A new somatostatin analog with improved target tissue uptake.

Radiolabeled somatostatin analogs have demonstrated potential as cancer therapeutic agents. Many of these agents are based on the analog octreotide (OC). Recently it has been shown that substitution of a tyrosine for phenylalanine in the 3-position and changing the C-terminus from an alcohol to an acid improves the targeting of somatostatin-rich tissues. The compound, 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid-Tyr3-octreotate (TETA-Y3-TATE), was synthesized and radiolabeled with 64Cu. The receptor binding properties of 64Cu-TETA-Y3-TATE showed an estimated Kd value of 549 pM in somatostatin receptor-positive CA20948 tissue membrane. High tumor uptake was observed in two animal tumor models. Tumor uptakes of 2.37 %ID/g in CA20948 tumor-bearing rats and 21.60 %ID/g in AR42J tumor-bearing SCID mice were observed at 1 h, compared with 1.09 %ID/g and 11.24 %ID/g for 64Cu-TETA-OC. Higher uptake in other somatostatin-receptor rich tissues was also observed, compared with 64Cu-TETA-OC. Positron emission tomography (PET) imaging with 64Cu-TETA-Y3-TATE in a baboon showed significant uptake in the pituitary and adrenals, and clearance through the kidneys. 64Cu-TETA-Y3-TATE, a new OC analog for binding somatostatin receptors, demonstrated significantly greater uptake in somatostatin-rich tissues in two tumor-bearing animal models, and demonstrated great potential as a radiopharmaceutical for imaging and therapy of somatostatin receptor-positive tissues.

Animals↗
PubMed1999

Insertion of EspD into epithelial target cell membranes by infecting enteropathogenic Escherichia coli.

Diffusely adhering Escherichia coli (DAEC) strains have been implicated in epidemiological studies as a cause of diarrhoea in children. However, the molecular interactions of these pathogens with target cells have remained largely obscure. We found that some DAEC strains contain homologues of the locus of enterocyte effacement (LEE) pathogenicity island and secrete EspA, EspB and EspD proteins necessary for the formation of the attaching and effacing (A/E) lesions. To characterize the function of the EspD protein further, we cloned and sequenced the espD genes of two DA-EPEC strains and compared their deduced amino-acid sequences with known EspD sequences. A pattern of two conserved transmembrane regions and one conserved coiled-coil region is predicted in EspD and also in the type III system secreted proteins YopB, PopB, IpaB and SipB of Yersinia, Pseudomonas, Shigella and Salmonella respectively. The EspD protein is inserted into a trypsin-sensitive location in the HeLa cell membrane at sites of bacterial contact, but is not translocated into the cytoplasm. Secretion of EspD increases upon contact with host cells. We propose that the membrane-located EspD protein is part of the translocation apparatus for Esp proteins into the target host cell performing functions similar to YopB in Yersinia.

Blotting, Western↗
PubMed1999

Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications.

Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates. The use of PDMS structures in microfluidics and biological applications is discussed. We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer. Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity. The latter is mostly limited by the mechanical stability of the replicated PDMS structures. As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells.

Biocompatible Materials↗
PubMed1999

Nonrestricted differential intoxication of cells by pertussis toxin.

After uptake and retrograde transport pertussis toxin acts by ADP-ribosylating alpha-Gi proteins. We show that uptake via many different receptor proteins followed by retrograde transport and intoxication is not restricted to a particular cell type. The efficiency of cellular intoxication, however, was found to be cell type dependent.

Adenosine Diphosphate Ribose↗
PubMed1999

Microfabrication in biology and medicine.

Microfabrication uses integrated-circuit manufacturing technology supplemented by its own processes to create objects with dimensions in the range of micrometers to millimeters. These objects can have miniature moving parts, stationary structures, or both. Microfabrication has been used for many applications in biology and medicine. These applications fall into four domains: tools for molecular biology and biochemistry, tools for cell biology, medical devices, and biosensors. Microfabricated device structures may provide significantly enhanced function with respect to a conventional device. Sometimes microfabrication can enable devices with novel capabilities. These enhancing and enabling qualities are conferred when microfabrication is used appropriately to address the right types of problems. Herein, we describe microfabrication technology and its application to biology and medicine. We detail several classes of advantages conferred by microfabrication and how these advantages have been used to date.

Animals↗
PubMed1999

Radiotherapy and dosimetry of 64Cu-TETA-Tyr3-octreotate in a somatostatin receptor-positive, tumor-bearing rat model.

64Cu [T1/2 = 12.8 h; beta+ = 0.655 MeV (19%); beta- = 0.573 MeV (40%)] has shown promise as a radioisotope for targeted radiotherapy. It has been demonstrated previously that the somatostatin analogue 64Cu-TETA-octreotide (64Cu-TETA-OC, where TETA is 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid) significantly inhibited the growth of somatostatin receptor-positive CA20948 rat pancreatic tumors in Lewis rats (C. J. Anderson et al., J. Nucl. Med., 39: 1944-1951, 1998). In this study, we evaluated the radiotherapeutic efficacy of a new 64Cu-labeled somatostatin analogue, 64Cu-TETA-Tyr3-octreotate (64Cu-TETA-Y3-TATE), in CA20948 tumor-bearing rats. A single dose of 15 mCi (555 MBq) of 64Cu-TETA-Y3-TATE was shown to be more effective in reducing tumor burden than the same dose of 64Cu-TETA-OC. In multiple dose experiments, tumor-bearing rats were administered three doses of either 10 or 20 mCi (370 or 740 MBq) of 64Cu-TETA-Y3-TATE at 48-h intervals. Rats given 3x10 mCi (3x370 MBq) showed extended mean survival times compared with rats given a single dose; however, no complete regressions occurred. Complete regression of tumors was observed for all rats treated with 3x20 mCi (3x740 MBq), with no palpable tumors for approximately 10 days; moreover, the mean survival time of these rats was nearly twice that of controls. Toxicity was determined by physical appearance and hematological and enzyme analysis, which revealed no overt toxicity and only transient changes in blood and liver chemistry. Absorbed dose estimates showed the dose-limiting organ to be the kidneys. The radiotherapy results, along with absorbed dose estimates to target and clearance organs, confirm that 64Cu-labeled somatostatin analogues warrant continued consideration as agents for targeted radiotherapy.

Animals↗
PubMed1998

Comparison of (111)In-labeled somatostatin analogues for tumor scintigraphy and radionuclide therapy.

We evaluated the following (111)In-labeled somatostatin (SS) analogues (diethylenetriaminepentaacetic acid, DTPA; tetraazacyclododecanetetraacetic acid, DOTA): [DTPA0]octreotide, [DTPA0,Tyr3]octreotide, [DTPA0,D-Tyr1]octreotide, [DTPA0,Tyr3]octreotate [Thr(ol) in octreotide replaced with Thr], and [DOTA0,Tyr3]octreotide, in vitro and in vivo. In vitro, all compounds showed high and specific binding to SS receptors in mouse pituitary AtT20 tumor cell membranes, and IC50s were in the nanomolar range. Furthermore, all compounds showed specific internalization in rat pancreatic tumor cells; uptake of [(111)In-DTPA0,Tyr3]octreotate was the highest of the compounds tested, and that of [(111)In-DTPA0,D-Tyr1]octreotide was the lowest. Biodistribution experiments in rats showed that, 4, 24, and 48 h after injection of [(111)In-DTPA0,Tyr3]octreotide, [(111)In-DTPA0,Tyr3]octreotate, and [(111)In-DOTA0,Tyr3]octreotide, radioactivity in the octreotide-binding, receptor-expressing tissues and tumor-to-blood ratios were significantly higher than those after injection of [(111)In-DTPA0]octreotide. Uptake of [(111)In-DTPA0,Tyr3]octreotate in the target organs was also, in vivo, the highest of the radiolabeled peptides tested, whereas that of [(111)In-DTPA0,D-Tyr1]octreotide was the lowest. Uptake of [(111)In-DTPA0,Tyr3]octreotide, [(111)In-DTPA0,Tyr3]octreotate, and [(111)In-DOTA0,Tyr3]octreotide in target tissues was blocked by >90% by 0.5 mg of unlabeled octreotide, indicating specific binding to the octreotide receptors. Blockade of [(111)In-DTPA0,D-Tyr1]octreotide was >70%. In conclusion, radiolabeled [DTPA0,Tyr3]octreotide and, especially, [DTPA0,Tyr3]octreotate and their DOTA-coupled counterparts are most promising for scintigraphy and radionuclide therapy of SS receptor-positive tumors in humans.

Animals↗
PubMed1998

FID-based lung MRI at 0.5 T: theoretical considerations and practical implications.

Pulse sequences based on FID signals and projection reconstruction (PR) were investigated for lung MRI at 0.5 T and evaluated for artifacts caused by: (1) k-space mismapping due to either delay or distortion of the readout gradient waveform, (2) cardiac motion and pulsatile flow, and (3) respiratory motion. Nonstructured artifacts were described, simulated, and experimentally confirmed for the first time. Nonstructured artifacts did not impair the demonstration of structures of high signal-to-noise ratio (SNR) but generated quantitative errors in the image intensity analysis over the lung parenchyma. The use of FID-based PR techniques for lung MRI is not justified at 0.5 T.

Artifacts↗