Aptamer-Based Affinity Chromatography for Protein Extraction and Purification.

Aptamers are oligonucleotide molecules able to recognize very specifically proteins. Among the possible applications, aptamers have been used for affinity chromatography with effective results and advantages over most advanced protein separation technologies. This chapter first discusses the context of the affinity chromatography with aptamer ligands. With the adaptation of SELEX, the chemical modifications of aptamers to comply with the covalent coupling and the separation process are then extensively presented. A focus is then made about the most important applications for protein separation with real-life examples and the comparison with immunoaffinity chromatography. In spite of well-advanced demonstrations and the extraordinary potential developments, a significant optimization work is still due to deserve large-scale applications with all necessary validations. Graphical Abstract Aptamer-protein complexes by X-ray crystallography.

Comparison between small bowel manometric patterns and full-thickness biopsy histopathology in severe intestinal dysmotility.

BACKGROUND: Intestinal manometry is the current standard for direct evaluation of small bowel dysmotility. Patients with abnormal motility can either be diagnosed of pseudo-obstruction when there are radiological findings mimicking mechanical intestinal obstruction or of enteric dysmotility when these findings are absent. The aim of the present study was to prospectively compare small bowel manometric abnormalities with histopathological findings in intestinal full-thickness biopsies in patients with severe dysmotility disorders. METHODS: We investigated 38 patients with intestinal manometry and a subsequent full-thickness intestinal biopsy. Manometric recordings were read by 4 investigators and a diagnostic consensus was obtained in 35 patients. Histopathological analysis, including specific immunohistochemical techniques of small bowel biopsies was performed and compared to manometric readings. KEY RESULTS: Patients with abnormal intestinal manometry had abnormal histopathological findings in 73% of cases. However, manometric patterns did not match with the specific neuromuscular abnormalities. Among patients with a neuropathic manometry pattern and abnormal histopathology, only 23% had an enteric neuropathy, whereas 62% had neuromuscular inflammation, and 15% an enteric myopathy. On the other hand, patients with a myopathic manometry pattern all had abnormal histopathology, however, none of them with signs of enteric myopathy. CONCLUSION & INFERENCES: Small bowel dysmotility detected by intestinal manometry is often associated with abnormal neuromuscular findings in full-thickness biopsies. However, there is no correlation between the specific manometric patterns and the histopathological findings.

Cerebral Mitochondrial Microangiopathy Leads to Leukoencephalopathy in Mitochondrial Neurogastrointestinal Encephalopathy.

BACKGROUND AND PURPOSE: Mitochondrial neurogastrointestinal encephalopathy is a rare disorder due to recessive mutations in the thymidine phosphorylase gene, encoding thymidine phosphorylase protein required for mitochondrial DNA replication. Clinical manifestations include gastrointestinal dysmotility and diffuse asymptomatic leukoencephalopathy. This study aimed to elucidate the mechanisms underlying brain leukoencephalopathy in patients with mitochondrial neurogastrointestinal encephalopathy by correlating multimodal neuroradiologic features to postmortem pathology. MATERIALS AND METHODS: Seven patients underwent brain MR imaging, including single-voxel proton MR spectroscopy and diffusion imaging. Absolute concentrations of metabolites calculated by acquiring unsuppressed water spectra at multiple TEs, along with diffusion metrics based on the tensor model, were compared with those of healthy controls using unpaired t tests in multiple white matters regions. Brain postmortem histologic, immunohistochemical, and molecular analyses were performed in 1 patient. RESULTS: All patients showed bilateral and nearly symmetric cerebral white matter hyperintensities on T2-weighted images, extending to the cerebellar white matter and brain stem in 4. White matter, N-acetylaspartate, creatine, and choline concentrations were significantly reduced compared with those in controls, with a prominent increase in the radial water diffusivity component. At postmortem examination, severe fibrosis of brain vessel smooth muscle was evident, along with mitochondrial DNA replication depletion in brain and vascular smooth-muscle and endothelial cells, without neuronal loss, myelin damage, or gliosis. Prominent periependymal cytochrome C oxidase deficiency was also observed. CONCLUSIONS: Vascular functional and histologic alterations account for leukoencephalopathy in mitochondrial neurogastrointestinal encephalopathy. Thymidine toxicity and mitochondrial DNA replication depletion may induce microangiopathy and blood-brain-barrier dysfunction, leading to increased water content in the white matter. Periependymal cytochrome C oxidase deficiency could explain prominent periventricular impairment.

INPP4B overexpression and c-KIT downregulation in human achalasia.

BACKGROUND: Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasia patients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.

A new general approach to purify proteins from complex mixtures.

The selection of chromatography media and their sequential use represent a major difficulty to isolate a single protein from very crude protein extracts. The process described here consists of two main steps: (i) a rational selection of few media from a relatively large collection and (ii) the definition of the sequence of columns to get the best purity of the target protein. From the first step, one sorbent is selected for its properties to capture the protein to purify, regardless whether other protein impurities are also co-adsorbed; then 5-7 other complementary sorbents are identified to remove impurities but without interacting with the target protein under the same buffering conditions. The second step consists in superimposing sorbents under a cascade manner with the sorbent in charge to capture the target protein located in the last position. Non-adsorbed proteins are eliminated in the flowthrough; other impurities are progressively removed by the sorbent sequence and the target protein is finally desorbed and isolated from the last sorbent using an optimized gradient. All operations are performed with a single adsorption buffer for all columns and all monitoring performed by means of mass spectrometry associated with ProteinChip arrays and polyacrylamide gel electrophoresis. Examples of protein isolation/identification from human serum are described namely thyroxin-binding proteins and transferrin. The first is isolated thanks to a series of dye chromatography media, the second (transferrin) using current chromatographic media. In both cases the target proteins were purified at a level estimated of about 95% and 85%, respectively. Isolated proteins were pure enough for the purpose of formal identification by either peptide fingerprinting or sequencing.

Contribution of solid-phase hexapeptide ligand libraries to the repertoire of human bile proteins.

Proteins in bile may have important physiological functions and serve as disease biomarkers. Here, the protein composition of human gallbladder bile was analyzed using a recently described chromatography-like technology capable to enhance the signal of low-abundance species. First, proteins present in bile fluid were treated with immobilized peptide ligand libraries to concentrate dilute and very dilute species while concomitantly diluting the high-abundance proteins. The analysis of resulting protein mixture was then performed using LC-MS/MS after having classically separated proteins by a mini preparative gel electrophoresis. Overall 222 gene products were found; 143 of them were not reported before in proteomics studies. Ligand libraries by themselves contributed to find 81 new gene products distributed throughout different categories. The described chromatographic approach provides a significant contribution to the bile protein repertoire and opens new perspectives for the discovery of markers for specific biliary tract diseases.

Fatty acid composition of chicken breast meat is dependent on genotype-related variation of FADS1 and FADS2 gene expression and desaturating activity.

In Western countries the dietary guidance emphasizes the need to decrease the intake of saturated fatty acids and to replace them with polyunsaturated fatty acids (PUFA), particularly long chain n-3 PUFA (LC-PUFA). The production of poultry meat having a lower fat content and healthier fatty acid (FA) profile is a hot topic for the poultry industry, and the possibility to identify genotypes able to produce meat with a higher LC-PUFA content deserves attention. The aims of the present study were to evidence in chicken (i) a genotype-related different expression of the desaturating enzymes delta-6 (Δ6, EC 1.14.99.25), delta-5 (Δ5, EC 1.14.19.) and delta-9 (Δ9, EC 1.14.19.1); (ii) the impact of the hypothesized different expression on the meat FA composition; (iii) the distribution of desaturase products in the different lipid classes. Slow (SG), medium (MG) and fast (FG) growing chickens fed the same diet were evaluated either for the relative expression of FADS1, FADS2 and SCD1 genes in liver (by q-PCR), or for the FA composition of breast meat. MG and particularly SG birds showed a greater expression of FADS2 and FADS1 genes, a higher Δ6 and Δ5 activity (estimated using desaturase indices), and consequently a higher LC-PUFA content in the breast meat than FG birds. The relationship between genotype and desaturating ability was demonstrated, with a significant impact on the PUFA content of breast meat. Due to the high consumption rate of avian meat, the identification of the best genotypes for meat production could represent an important goal not only for the food industry, but also for the improvement of human nutrition.

Contribution of autonomic neuropathy to reduced plasma adrenaline responses to hypoglycemia in IDDM: evidence for a nonselective defect.

To determine the contribution of clinically overt diabetic autonomic neuropathy (DAN) to reduced plasma adrenaline responses to hypoglycemia in IDDM and to establish its selectivity for hypoglycemia, we studied 17 IDDM patients (7 without DAN [DAN-] and 10 with DAN [DAN+]), of whom 5 had and 5 did not have postural hypotension (DAN+PH+ and DAN+PH-, respectively), and 8 nondiabetic subjects on 2 different occasions, i.e., clamped hypoglycemia (steps from 5.0 to 2.2 mmol/l plasma glucose) and 30-min steady-state exercise at 55% V(O[2max]). Recent antecedent hypoglycemia was meticulously prevented before the studies to exclude hypoglycemia as a cause of reduced responses of adrenaline to hypoglycemia. In DAN- patients, maximal responses of adrenaline to hypoglycemia were reduced (2.44 +/- 0.58 nmol/l vs. 4.9 +/- 0.54 nmol/l in nondiabetic patients) (P < 0.05). In DAN+, adrenaline responses initiated at a lower plasma glucose and were lower than in DAN- (DAN+PH-, 1.06 +/- 0.38 nmol/l; DAN+PH+, 0.84 +/- 0.27 nmol/l; P < 0.001, but NS between PH- and PH+). In response to exercise, adrenaline increased less in DAN- (0.89 +/- 0.11 nmol/l) patients than in nondiabetic subjects (1.19 +/- 0.14 nmol/l; NS) and only to 0.36 +/- 0.07 nmol/l in DAN+PH- and 0.23 +/- 0.09 nmol/l in DAN+PH+ (P < 0.001 vs. DAN- and nondiabetic subjects). These results were confirmed when nondiabetic and DAN- subjects repeated the exercise at 60 watts (35 and 41% of V(O[2max]), respectively), i.e., at the same absolute workload of DAN+ patients. Thus, DAN (both PH+ and PH-) contributes to reduced responses of adrenaline to hypoglycemia independently of recent antecedent hypoglycemia. The adrenaline defect in DAN is not selective for hypoglycemia.

A simplified monobuffer multidimensional chromatography for high-throughput proteome fractionation.

The complexity of the human serum proteome is attributed to both a large dynamic range of protein abundance, as much as 10 orders of magnitude, and a disproportionate few dozens of proteins representing as much as 99% of the total protein content. These characteristics make it beneficial to use a pre-fractionation step prior to any high-resolution analysis, such as mass spectrometry. The present method describes a unimodal multidimensional chromatography concept to rapidly achieve an effective fractionation of human serum that is directly amenable with surface-enhanced laser desorption/ionization (SELDI)-based mass spectrometry. This method is based on the use of a column composed of a superimposed sequence of sorbents. The assembly is first equilibrated with a single binding buffer and then loaded with the whole crude sample. As the sample crosses the different adsorbent layers proteins within are sequentially trapped according to the complementary properties vis-a-vis of the sorbent. Once the loading and capturing is achieved, the sequence of columns is disassembled and each column, containing different complement of proteins is eluted separately in a single step and under optimal elution conditions. When compared to classical single-chemistry fractionation based on, for example, anion-exchange and pH stepwise elution, the new proposed approach shows much lower protein overlap between fractions, and therefore, greater resolution. This results in a larger number of detectable species, and therefore, reinforces the power of discovery of new biomarkers. A significantly higher sensitivity for low-abundance species was additionally found as evidenced by spiking trials.

Activation of µ opioid receptors modulates inflammation in acute experimental colitis.

BACKGROUND: µ opioid receptors (µORs) are expressed by neurons and inflammatory cells, and mediate immune response. We tested whether activation of peripheral µORs ameliorates the acute and delayed phase of colitis. METHODS: C57BL/6J mice were treated with 3% dextran sodium sulfate (DSS) in water, 5 days with or without the peripherally acting µOR agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or with DAMGO+µOR antagonist at day 2-5, then euthanized. Other mice received DSS followed by water for 4 weeks, or DSS with DAMGO starting at day 2 of DSS for 2 or 3 weeks followed by water, then euthanized at 4 weeks. Disease activity index (DAI), histological damage, and myeloperoxidase assay (MPO), as index of neutrophil infiltration, were evaluated. Cytokines and µOR mRNAs were measured with RT-PCR, and nuclear factor-kB (NF-kB), the antiapoptotic factor Bcl-xL, and caspase 3 and 7 with Western blot. KEY RESULTS: DSS induced acute colitis with elevated DAI, tissue damage, apoptosis and increased MPO, cytokines, µOR mRNA, and NF-kB. DAMGO significantly reduced DAI, inflammatory indexes, cytokines, caspases, and NF-kB, and upregulated Bcl-xL, effects prevented by µOR antagonist. In DSS mice plus 4 weeks of water, DAI, NF-kB, and µOR were normal, whereas MPO, histological damage, and cytokines were still elevated; DAMGO did not reduce inflammation, and did not upregulate Bcl-xL. CONCLUSIONS & INFERENCES: µOR activation ameliorated the acute but not the delayed phase of DSS colitis by reducing cytokines, likely through activation of the antiapoptotic factor, Bcl-xL, and suppression of NF-kB, a potentiator of inflammation.

Double-blind crossover trial of single vs. divided dose of metoclopramide in a combined regimen for treatment of cisplatin-induced emesis.

In a double-blind crossover antiemetic study in cisplatin-treated cancer patients, metoclopramide 4 mg/kg as a single intravenous dose (regimen A) was compared with 3 mg/kg in two doses (regimen B). In both regimens, metoclopramide was combined with dexamethasone and diphenhydramine. 65 consecutive, chemotherapy-naïve inpatients (45 males and 20 females) treated with high doses (at least 50 mg/m2) of cisplatin entered the study and 54 completed both treatments. Complete protection from vomiting and nausea, mean number of emetic episodes, mean maximum intensity of nausea and mean duration of emesis or nausea were similar with the two antiemetic regimens. 23 patients (43%) did not express a treatment preference, while 16 (30%) preferred regimen B and 15 (28%) preferred regimen A. Side-effects were similar with the two metoclopramide schedules. A combined antiemetic regimen of a single high dose of metoclopramide (4 mg/kg) can preserve efficacy and tolerability and thus should be preferred.

Cell affinity chromatography with ligands immobilized through cleavable mercury-sulfur bonds.

A new methodology for cell separation by affinity chromatography is described. We have conjugated the organomercurial mersalyl to trisacryl beads bearing primary amino groups. Thiolated ligands can be immobilized on this matrix through cleavable Hg-S bonds. Two model studies of cell separation are reported: (i) concanavalin A thiolated with N-succinimidyl-3-(2-pyridyldithio)-propionate and immobilized on mersalyl-trisacryl; mouse thymocytes bound to Con A-mersalyl-trisacryl were eluted from the support by short thiol treatment which preserved cell viability; (ii) anti-dinitrophenyl antibodies modified with S-acetyl-mercaptosuccinic anhydride and immobilized on mersalyl-trisacryl; sheep erythrocytes, previously labelled with trinitrobenzene sulfonic acid, bound to this support and were easily recovered by thiol treatment without hemolysis. This methodology should overcome difficulties frequently encountered in cell affinity chromatography. Cell support multivalent interactions or high affinity of cell-ligand bindings often require drastic elution conditions which prevent viable cell recovery.

Features of thrombi and diagnostic accuracy of impedance plethysmography in symptomatic and asymptomatic deep vein thrombosis.

Impedance plethysmography (IPG) has high sensitivity and specificity in patients with symptomatic deep vein thrombosis (DVT) while it fails to detect asymptomatic DVT. The aim of this study was to determine whether the features of thrombi such as location, size and occlusiveness could explain the different accuracy of IPG in symptomatic and asymptomatic DVT patients. One-hundred and seventeen consecutive outpatients with a clinical suspicion of DVT and 246 consecutive patients undergoing hip surgery were admitted to the study. In symptomatic patients IPG was performed on the day of referral, followed by venography, while in asymptomatic patients IPG was performed as a surveillance programme, followed by bilateral venography. A venography proved DVT was observed in 37% of the symptomatic patients and 34% of the asymptomatic limbs. A significantly higher proportion of proximal DVTs was found in symptomatic patients than in asymptomatic patients (78% vs 46%; p = 0.001). The mean Marder score, taken as an index of thrombus size, was significantly higher in symptomatic patients than in asymptomatic patients (19.0 vs 9.6; p = 0.0001). A significantly higher proportion of occlusive DVTs was observed in symptomatic than in asymptomatic patients (69% vs 36%; p = 0.001). We conclude that the unsatisfactory diagnostic accuracy of IPG in asymptomatic DVT is due to the high prevalence of distal, small and non occlusive thrombi. Such thrombi are unlikely to cause a critical obstruction of the venous outflow and therefore to produce a positive IPG.

Correlation between clinical and morphologic findings in unstable angina.

This study was undertaken to verify the hypothesis that the discrepant findings in published reports on the prevalence of thrombus in unstable angina depend on the inclusion of different clinical subsets in the various studies. We therefore correlated the clinical characteristics of patients included under the label of unstable angina with the morphologic features assessed by coronary angiography and intravascular ultrasound, and with histopathologic findings of atherectomy specimens. Fifty-eight patients with unstable angina (class B of the Braunwald classification) undergoing coronary arteriography followed by either coronary angioplasty (n = 20) or directional coronary atherectomy (n = 38) were studied. Fifteen patients were in class IB and 43 were in class II to IIIB. Among these 43 patients with angina at rest, 28 had ST-segment elevation during pain, and 15 had ST-segment depression, and 26 developed negative T waves on the baseline electrocardiogram (ECG) as a result of prolonged or repeated episodes of resting chest pain. Intravascular ultrasound examination of the culprit lesion was performed in 43 patients before the interventional procedure, and histopathologic analysis of atherectomy specimens was performed in 38 patients. Complex lesion morphology by angiography was observed in 31 patients (53%) without any significant relation to various clinical subsets. Patients in Braunwald class IB had more calcific plaques than patients in class II to IIIB (p < 0.001). Among patients with angina at rest, those with negative T waves on the baseline ECG, as well as those with transient ST elevation during pain, had a significantly higher incidence of noncalcific lesions (p = 0.001 for both). Analysis of atherectomy specimens revealed acute coronary lesions (thrombus and/or intraplaque hemorrhage) in 18 patients (47%). The incidence of acute coronary lesions was significantly higher in patients with than without negative T waves on the baseline ECG (p = 0.005), and increased further when negative T waves were combined with ST elevation during pain (p = 0.001). Multivariate analysis revealed that the presence orf negative T waves on the baseline ECG was the only explanatory variable related to the presence of acute coronary lesions by histology (p = 0.03). Patient subsets included in the broad spectrum of unstable angina have different morphologic features and incidence of acute coronary lesions by histology. These data provide an explanation for the discrepant findings in published reports on the relevance of thrombus formation in the pathogenesis of unstable angina.

Expression of neutrophil and monocyte CD11B/CD18 adhesion molecules at different sites of the coronary tree in unstable angina pectoris.

To assess the site of leukocyte activation in unstable angina, the expression of neutrophil and monocyte CD11B/CD18 adhesion molecules in 26 patients was measured from blood samples taken from the coronary ostium, the coronary sinus, and the coronary artery just distal to the culprit lesion (postobstructive chamber). CD11B/CD18 adhesion molecules detected by direct immunofluorescence evaluated by flow cytometry were significantly higher in the coronary sinus blood than in both the coronary ostium and the postobstructive chamber blood, suggesting that leukocyte activation takes place at the microcirculatory interface with the injured myocardium, probably as the result of short but repeated episodes of myocardial ischemia.

Factors affecting the therapeutic choice in patients with multivessel coronary artery disease. The Studio Lombardo Angiografia Multivasali (SLAM) Study Group.

OBJECTIVE: To assess how clinical and angiographic findings are related to the decision to carry out coronary angioplasty (PTCA) or coronary bypass grafting in patients with multivessel coronary artery disease. DESIGN: Prospective survey carried out in 14 centres in the Lombardia region of Italy. PATIENTS: 1468 consecutive patients under going coronary arteriography for known or suspected ischaemic heart disease between May and October 1994, who were found to have multivessel coronary artery disease. MAIN OUTCOME MEASURES: Multivariate analysis was undertaken using stepwise logistic regression to identify the clinical and angiographic variables correlated with revascularisation (v medical treatment) in all of patients, and with surgery (v angioplasty) in the subset of revascularised patients. RESULTS: In all patients the clinical decision after coronary arteriography was made by physicians of each participating centre on the basis of their experience and clinical judgment: 53% of patients had bypass surgery, 28% had PTCA, and 19% continued medical treatment. The choice of a revascularisation procedure was directly related to a clinical diagnosis of unstable angina (P < < 0.001), the presence of left anterior descending artery disease (P < < 0.001), and to an ejection fraction > or = 40% (P < < 0.001), and inversely related to history of previous coronary bypass surgery (P < < 0.001). In revascularised patients, bypass surgery was the preferred treatment in patients with left anterior descending artery disease (P < < 0.001), three-vessel disease (P < < 0.001), and in those with at least one occluded vessel (P = 0.008). The choice of PTCA was significantly related to history of previous PTCA (P < < 0.001) or coronary bypass surgery (P < < 0.001), to a clinical diagnosis of non-Q wave myocardial infarction (P = 0.002), and to the possibility of implanting an intracoronary stent (P = 0.01). CONCLUSIONS: Bypass surgery is still the most widely used treatment for patients with multivessel coronary artery disease. This analysis provides a basis for comparison with future developments in the treatment of such patients. Further advancements in PTCA technology are needed to tilt the balance in favour of this less invasive procedure.

Effect of aspirin and dipyridamole treatment on prostacyclin production by human veins.

Patients admitted for surgical removal of varicose veins were treated in a blinded manner for 48 hours prior to surgery with either placebo, low-dose aspirin (25 mg twice daily), dipyridamole (150 mg twice daily) or both. Segments of vein excised at surgery were incubated with or without sodium arachidonate and subsequent prostacyclin (PGI2) production was measured without knowledge of treatment given. During the first 5 minute period of incubation in the presence of arachidonate, veins from dipyridamole-treated patients demonstrated increased (by 75%) arachidonate-stimulated PGI2 production compared to placebo-treated patients. By contrast, PGI2 production was reduced by 64% by aspirin treatment and 67% by aspirin plus dipyridamole compared to placebo-treated patients (p = less than 0.05). In unstimulated vein segments incubated in the absence of arachidonate, spontaneous PGI2 production during the first 5 minute incubation period was increased 32% following dipyridamole treatment but was unchanged following aspirin treatment. By contrast, unstimulated (spontaneous) PGI2 production in patients treated with aspirin plus dipyridamole was reduced by 57% (p = less than 0.05), compared to both placebo- and aspirin-treated patients, and by 71% (p = less than 0.05) compared to dipyridamole-treated patients. With repeated change of incubation medium, the ability of vein walls to produce PGI2 declined. This exhaustion was not prevented by drug treatment. However, drug effects between patient treatment groups were consistent over successive incubation periods. These results suggest that certain therapeutic benefits that might be achieved by enhancement of PGI2 production from vascular endothelium following dipyridamole treatment may be reduced by simultaneous aspirin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Trisacryl gelatin microspheres for therapeutic embolization, I: development and in vitro evaluation.

PURPOSE: To develop a precisely calibrated, perfectly spherical, stainable, soft, and implantable but nonresorbable particulate embolization material. METHODS: Calibrated particles with a trisacryl gelatin polymer core and hydrophilic surface characteristics were obtained by reversed emulsion synthesis followed by application of a wet-sieving technique. Particles were suspended in saline, bottled, and sterilized. Quality control included analysis of particle diameters before and after sieving and of suspension sterility and apyrogenicity. Particles were subsequently tested to ascertain their compatibility with commercially available microcatheters. RESULTS: The resulting embolization material consisted of spherical, stainable microspheres of medical grade with diameters ranging from 130 microns to 1200 microns. Sieving the suspension produced particle groups of homogeneous size (accuracy, +/- 20-100 microns). At injection, the particles showed no tendency to build aggregates or to obstruct the microcatheters. CONCLUSION: Precisely calibrated and easy-to-use microspheres were obtained that satisfied the biomedical requirements for implantation as an embolization material.

Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies.

Efficient harvest and recovery of high-purity monoclonal antibodies was achieved using hydrophobic charge induction chromatography (HCIC). Both simple and complex feedstocks were studied, including protein-free cell culture supernatant and the clarified/concentrated milk of transgenic goats. Viral clearance studies demonstrated a 4-log reduction of MVM virus (minute virus of mice), along with substantial reduction of DNA content. Sorbent characterization studies confirmed that HCIC is based on the pH-dependent behavior of a dual-mode, ionizable ligand. Binding, based on hydrophobic interaction, was achieved under near-physiological conditions, and in the absence of lyotropic salt. Desorption was accomplished under mild conditions--pH 4.0. At this pH, both ligand and antibody carry a net positive charge, and desorption occurs on the basis of electrostatic charge repulsion. pH-based control of chromatographic function was demonstrated. Chromatography on this antibody-selective HCIC sorbent was evaluated as a cost-effective, process-compatible alternative to affinity chromatography protein A sorbents.

2-Mercapto-5-benzimidazolesulfonic acid: an effective multimodal ligand for the separation of antibodies.

The report describes the use of 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as a ligand for the separation of antibodies by chromatography. The ligand shows a relatively specific adsorption property for antibodies from very crude biologicals at pH 5.0-5.5. At this pH range most of other proteins do not interact with the resin especially when the ionic strength is similar to physiological conditions. Several characterization studies are described such as antibody adsorption in different conditions of ionic strength, pH and temperature. These properties are advantageously used to selectively capture antibodies from very crude feed stocks without dilution or addition of lyotropic salts. Demonstration was made that the adsorption mechanism is neither based on ion exchange nor on hydrophobic associations, but rather as an assembly of a variety of properties of the ligand itself. Binding capacity in the described conditions ranges between 25 and 30 mg/mL of resin. The sorbent does not co-adsorb albumin (Alb) and seems compatible with a large variety of feedstocks. Quantitative antibody desorption occurs when the pH is raised above 8.5. The final purity of the antibody depends on the nature of the feedstock, and can reach levels of purity as high as 98%. Even with very crude biological liquids such as ascites fluids, cell culture supernatants and Chon fraction II + III from human plasma fractionation where the number of protein impurities is particularly large, immunoglobumins G (IgG) were separated at high purity level in a single step.