sepharose and Neoplasms

Seaweeds are some of the largest producers of biomass in the marine environment and are rich in bioactive compounds that are often used for human and animal health. Porphyran and carrageenan are natural compounds derived from red seaweeds. The former is a characteristic polysaccharide of

Topics: Carrageenan; Humans; Immunity; Neoplasms; Polysaccharides; Seaweed; Sepharose

The hazardous effects of current nanoparticle synthesis methods have steered researchers to focus on the development of newer environmentally friendly and green methods for synthesizing nanoparticles using nontoxic chemicals. The development of environmentally friendly methods of nanoparticle synthesis with different sizes and shapes is one of the pressing challenges for the current nanotechnology. Several novel green approaches for the synthesis of AuNPs have been explored using different natural sources, such as plants, algae, bacteria, and fungi. Among organisms, algae and blue-green algae are of particular interest for nanoparticle synthesis. Gold nanoparticles (AuNPs) have a range of applications in medicine, diagnostics, catalysis, and sensors because of their significant key roles in important fields. AuNPs have attracted a significant interest for use in a variety of applications. The widespread use of AuNPs can be accredited to a combination of optical, physical, and chemical properties as well as the miscellany of size, shape, and surface composition that has been adopted through green synthesis methods.

Topics: Antioxidants; Bacterial Infections; Catalysis; Cell Line, Tumor; Cyanobacteria; Fungi; Gold; Green Chemistry Technology; Humans; Metal Nanoparticles; Nanotechnology; Neoplasms; Plants; Polymers; Seaweed; Sepharose; Surface Properties

The recruitment of effector cells is one of the novel functions described for extracellular vesicles (EVs) that needs further study. For instance, cell recruitment by mesenchymal stromal cell derived-EVs (MSC-EVs) is one of the features by which MSC-EVs may induce regeneration and ameliorate tissue injury. On the other hand, increasing evidence suggests that cancer EVs play an important role in the preparation of the pre-metastatic niche (PMN) by recruiting their primary tumour cells. Understanding and measuring the potential of MSC-EVs or cancer-EVs to induce cell migration and recruitment is essential for cell-free therapeutic approaches and/or for a better knowledge of cancer metastasis, respectively. In this context, classical in vitro migration assays do not completely mimic the potential situation by which EVs exert their chemotactic capacity.. We adapted an agarose spot migration assay as an in vitro system to evaluate the cell recruitment capacity of locally delivered or localized EVs. Cell migration was tracked for 12 h or 48 h, respectively. Thereafter, endpoint migration images and time-lapse videos were analysed to quantify several parameters aiming to determine the migration of cells to either MSC-EV or pro-metastatic EV. The number of cells contained inside the agarose spots, the migration distance, the area occupied by cells, the directionality of the cell movement, and the Euclidean distance were measured. This multi-parametric evaluation revealed the potential of different MSC-EV preparations to recruit endothelial cells and to detect an enhanced recruitment capacity of highly metastatic PC3-derived EVs (PC3-EVs) compared to low-metastatic LNCaP-EVs in a tumour cell-specific manner.. Overall, this agarose spot migration assay may offer a diversity of measurements and migration settings not provided by classical migration assays and reveal its potential use in the EV field in two different contexts with recruitment in common: regeneration and cancer metastasis.

Topics: Chemotactic Factors; Endothelial Cells; Extracellular Vesicles; Humans; Neoplasms; Regenerative Medicine; Sepharose

Hydrogels of varying complexity are routinely used as scaffolds and 3D structures for in vitro tumor models to increase physiological relevance within pre-clinical cancer research. Relatively simple hydrogels such as agarose are well characterised, meanwhile biomimetic gels containing collagen and fibrin(ogen) have been studied to a much lesser extent. In this study, hydrogels mimicking the biophysical characteristics of liver cancer progression were investigated in terms of their UV-properties and influence on diffusion coefficients of different substances. UV-imaging technology was used to both visualize and quantify the diffusion process in a simple and rapid way. In general, agarose gel diffusion agreed well with predictions using the Stokes-Einstein equation meanwhile the biomimetic gels reduced diffusion coefficients by up to 70%. For doxorubicin, spatio-temporal tissue concentration modelling was used to translate in vitro diffusion to the more clinical context of tumor penetration in a solid liver tumor supplied by arterial blood.

Topics: Biomimetics; Collagen; Diffusion; Humans; Hydrogels; Neoplasms; Sepharose

The extracellular matrix (ECM) is an essential component of the tumor microenvironment. It plays a critical role in regulating cell-cell and cell-matrix interactions. However, there is lack of systematic and comparative studies on different widely-used ECM mimicking hydrogels and their properties, making the selection of suitable hydrogels for mimicking different in vivo conditions quite random. This study systematically evaluates the biophysical attributes of three widely used natural hydrogels (Matrigel, collagen gel and agarose gel) including complex modulus, loss tangent, diffusive permeability and pore size. A new and facile method was developed combining Critical Point Drying, Scanning Electron Microscopy imaging and a MATLAB image processing program (CSM method) for the characterization of hydrogel microstructures. This CSM method allows accurate measurement of the hydrogel pore size down to nanometer resolution. Furthermore, a microfluidic device was implemented to measure the hydrogel permeability (P

Topics: Biophysical Phenomena; Collagen; Extracellular Matrix; Humans; Hydrogels; Neoplasms; Sepharose; Tumor Microenvironment

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Topics: Humans; Imaging, Three-Dimensional; Neoplasms; Sepharose; Spheroids, Cellular

Radiofrequency ablation (RFA) is a minimally invasive form of thermotherapy with great potential in cancer care, having the capability of selectively ablating tumoral masses with a surface area of several cm

Topics: Catheter Ablation; Humans; Liver; Neoplasms; Optical Fibers; Radiofrequency Ablation; Sepharose

The principal cause of cancer deaths is the residual disease, which eventually results in metastases. Certain metastases are induced by disseminated dormancy-capable single cancer cells that can reside within the body undetected for months to years. Awakening of the dormant cells starts a cascade resulting in the patient's demise. Despite its established clinical significance, dormancy research and its clinical translation have been hindered by lack of in vitro models that can identify, isolate, and analyze dormancy-capable cells. We have previously shown that immobilization of cells in a stiff microenvironment induces dormancy in dormancy-capable cell lines. In this communication, we present a novel biomaterial and an in vitro immobilization method to isolate, analyze, and efficiently recover dormancy-capable cancer cells. MCF-7, MDA-MB-231, and MDA-MB-468 cells were individually coated with agarose using a microfluidic flow-focusing device. Coated cells were then immobilized in a rigid and porous silica gel. Dormancy induction by this process was validated by decreased Ki-67 expression, increased p38/ERK activity ratio, and reduced expression of CDK-2, cyclins D1, and E1. We showed that we can reliably and repeatedly induce dormancy in dormancy-capable MCF-7 cells and enhance the dormancy-capable sub-population in MDA-MB-231 cells. As expected, dormancy-resistant MDA-MB-468 cells did not survive immobilization. The dormant cells could be awakened on demand, by digesting the agarose gel in situ, and efficiently recovered by magnetically separating the silica gel, making the cells available for downstream analysis and testing. The awakened cells were shown to regain motility immediately, proliferating, and migrating normally.

Topics: Biocompatible Materials; Humans; MCF-7 Cells; Neoplasms; Sepharose; Silicon Dioxide

In recent years, 3D culture of tumor spheroids has managed to revolutionize cancer research and drug discovery. 2D monolayer cells grown in cell culture flasks undergo radical changes in cell behavior, structure, and function owing to varying environmental cues and are unable to provide predictive data for preclinical evaluation. 3D tumor spheroids can better recapitulate tumor architecture, cell-cell and cell-matrix connectivity, and the tissue complexity of tumors grown in animal models. However, many of the existing techniques to culture 3D spheroids are time-consuming and ineffective and produce irregular-shaped spheroids that cannot be easily incorporated in biological assays. The set of protocols described herein makes use of a commercial hair brush as a template to create concave micro-well impressions in agarose. This technique is easy, inexpensive, and adaptable and also has the ability to produce uniform, homogenous cancer spheroids, with large diameter (∼1000 μm) and thickness (∼250 μm), within 24 to 48 hr after cell seeding. The 3D spheroids produced using the agarose micro-well platform function as an excellent 3D in vitro model for understanding the extent of penetration, uptake, and distribution of targeted cargos such as a diagnostic or therapeutic agents for identification and treatment of cancer. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of agarose micro-well scaffold for growing tumor spheroids using a commercial hair brush Basic Protocol 2: Formation of homogenous tumor spheroids in agarose micro-well platform Basic Protocol 3: Assessing viability of 3D tumor spheroids grown in agarose micro-wells using confocal microscopy Basic Protocol 4: Analyzing uptake and penetration of targeted fluorescent bioconjugate in 3D tumor spheroids using two-photon imaging.

Topics: Animals; Cell Culture Techniques; Drug Discovery; Neoplasms; Sepharose; Spheroids, Cellular

Molecular analysis of DNA samples with limited quantities can be challenging. Repeatedly sequencing the original DNA molecules from a given sample would overcome many issues related to accurate genetic analysis and mitigate issues with processing small amounts of DNA analyte. Moreover, an iterative, replicated analysis of the same DNA molecule has the potential to improve genetic characterization. Herein, we demonstrate that the use of "click"-based attachment of DNA sequencing libraries onto an agarose bead support enables repetitive primer extension assays for specific genomic DNA targets such as gene exons. We validated the performance of this assay for evaluating specific genetic alterations in both normal and cancer reference standard DNA samples. We demonstrate the stability of conjugated DNA libraries and related sequencing results over the course of independent serial assays spanning several months from the same set of samples. Finally, we finally applied this method to DNA derived from a tumor sample and demonstrated improved mutation detection accuracy.

Topics: Cell Line, Tumor; Click Chemistry; Cycloaddition Reaction; DNA, Neoplasm; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Mutation; Neoplasms; Proof of Concept Study; Sepharose

OBJECTIVE To characterize spatial release of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CI-CSH) beads by use of an agarose tissue phantom. SAMPLE 3-mm-diameter beads (n = 60) containing 4.6 mg of carboplatin (2.4 mg of platinum)/bead. PROCEDURES 18 L of 1% agarose was prepared and poured into 36 containers (10 × 10 × 10 cm), each of which was filled half full (0.5 L/container). After the agarose solidified, 1, 3, 6, or 10 CI-CSH beads were placed on the agar in defined patterns. An additional 36 blocks of agar (0.5 L/block) were placed atop the beads, positioning the beads in the center of 1 L of agar. The experiment was replicated 3 times for each bead pattern for 24, 48, and 72 hours. At these times, representative agarose blocks were sectioned in the x-, y-, and z-planes and labeled in accordance with their positions in shells radiating 1, 2, 3, 4, and 5 cm from the center of the blocks. Agarose from each shell was homogenized, and a sample was submitted for platinum analysis by use of inductively coupled plasma-mass spectroscopy. RESULTS Platinum diffused from CI-CSH beads at predicted anticancer cytotoxic concentrations for 2 to 5 cm. CONCLUSIONS AND CLINICAL RELEVANCE Results provided information regarding the spatial distribution of platinum expected to occur in vivo. Agarose may be used as a diffusion model, mimicking the characteristics of subcutaneous tissues. Measured platinum concentrations might be used to guide patterns for implantation of CI-CSH beads in animals with susceptible neoplasms.

Topics: Animals; Calcium Sulfate; Carboplatin; Dogs; Drug Screening Assays, Antitumor; Gelatin; Inhibitory Concentration 50; Mass Spectrometry; Microspheres; Neoplasms; Phantoms, Imaging; Platinum; Sepharose; Time Factors

Barcodes-based suspension array have for demonstrated values in multiplex assay of tumor markers. Photonic barcodes which are encoded by their characteristic reflection peaks are the important supports for suspension array due to their stable code, low fluorescent background and high surface-volume ratio. Attempts to develop this technology tend to improve the function of the photonic barcodes. Here, we present a new type of hybrid hydrogel photonic barcodes for efficient multiplex assays. This photonic barcodes are hybrid inverse opal hydrogel composed of poly(ethylene glycol) diacrylate (PEG-DA) and agarose. The polymerized PEG-DA hydrogel could guarantee the stabilities of the inverse opal structure and its resultant code, while the agarose could offer active chemical groups for the probe immobilization and homogeneous water surrounding for the bioassay. In addition, the interconnected pores inverse opal structure could provide channels for biomolecules diffusing and reaction into the voids of barcodes. These features imparted the hybrid hydrogel photonic barcodes with limits of detection (LOD) of 0.78ng/mL for carcinoembryonic antigen (CEA) and 0.21ng/mL for α-fetoprotein (AFP), respectively. It was also demonstrated that the proposed barcodes showed acceptable accuracy and detection reproducibility, and the results were in acceptable agreement with those from common clinic method for the detections of practical clinical samples. Thus, our technique provides a new platform for simultaneous multiplex immunoassay.

Topics: alpha-Fetoproteins; Antibodies, Immobilized; Biomarkers, Tumor; Biosensing Techniques; Carcinoembryonic Antigen; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Immunoassay; Limit of Detection; Neoplasms; Polyethylene Glycols; Reproducibility of Results; Sepharose

We synthesized agarose-polycaprolactone (Agr-PCL) bicomponent and Agr-polyethylene glycol-PCL (Agr-PEG-PCL) tricomponent amphiphilic co-network (APCN) gels by the sequential nucleophilic substitution reaction between amine-functionalized Agr and activated halide terminated PCL or PCL-b-PEG-b-PCL copolymer for the sustained and localized delivery of hydrophilic and hydrophobic drugs. The biodegradability of the APCNs was confirmed using lipase and by hydrolytic degradation. These APCN gels displayed good cytocompatibility and blood compatibility. Importantly, these APCN gels exhibited remarkably high drug loading capacity coupled with sustained and triggered release of both hydrophilic and hydrophobic drugs. PEG in the APCNs lowered the degree of phase separation and enhanced the mechanical property of the APCN gels. The drug loading capacity and the release kinetics were also strongly influenced by the presence of PEG, the nature of release medium, and the nature of the drug. Particularly, PEG in the APCN gels significantly enhanced the 5-fluorouracil loading capacity and lowered its release rate and burst release. Release kinetics of highly water-soluble gemcitabine hydrochloride and hydrophobic prednisolone acetate depended on the extent of water swelling of the APCN gels. Cytocompatibility/blood compatibility and pH and enzyme-triggered degradation together with sustained release of drugs show great promise for the use of these APCN gels in localized drug delivery and tissue engineering applications.

Topics: Drug Carriers; Drug Delivery Systems; Drug Liberation; Ethylene Glycols; Fluorouracil; Humans; Hydrogels; Hydrophobic and Hydrophilic Interactions; Neoplasms; Polyesters; Polyethylene Glycols; Sepharose

Magnetic iron oxide nanoparticles (MNPs) have been developed for magnetic fluid hyperthermia (MFH) cancer therapy, where cancer cells are treated through the heat generated by application of a high frequency magnetic field. This heat has also been proposed as a mechanism to trigger release of chemotherapy agents. In each of these cases, MNPs with optimal heating performance can be used to maximize therapeutic effect while minimizing the required dosage of MNPs. In this study, the heating efficiencies (or specific absorption rate, SAR) of two types of MNPs were evaluated experimentally and then predicted from their magnetic properties. MNPs were also incorporated in the core of poly(ethylene glycol-b-caprolactone) micelles, co-localized with rhodamine B fluorescent dye attached to polycaprolactone to monitor local, nanoscale temperatures during magnetic heating. Despite a relatively high SAR produced by these MNPs, no significant temperature rise beyond that observed in the bulk solution was measured by fluorescence in the core of the magnetic micelles. MNPs were also incorporated into a macro-scale agarose gel system that mimicked a tumor targeted by MNPs and surrounded by healthy tissues. The agarose-based tumor models showed that targeted MNPs can reach hyperthermia temperatures inside a tumor with a sufficient MNP concentration, while causing minimal temperature rise in the healthy tissue surrounding the tumor.

Topics: Animals; Ferric Compounds; Humans; Hyperthermia, Induced; Magnetic Field Therapy; Models, Biological; Nanoparticles; Neoplasms; Polyethylene Glycols; Sepharose

An antifungal peptide with a defensin-like sequence and exhibiting a molecular mass of 7.3kDa was purified from dried seeds of Phaseolus vulgaris 'Cloud Bean'. The isolation procedure entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography an Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Although the antifungal peptide was unadsorbed on DEAE-cellulose, it was adsorbed on both Affi-gel blue gel and SP-Sepharose. The antifungal peptide exerted antifungal activity against Mycosphaerella arachidicola with an IC(50) value of 1.8 μM. It was also active against Fusarium oxysporum with an IC(50) value of 2.2 μM. It had no inhibitory effect on HIV-1 reverse transcriptase when tested up to 100 μM. Proliferation of L1210 mouse leukemia cells and MBL2 lymphoma cells was inhibited by the antifungal peptide with an IC(50) of 10 μM and 40 μM, respectively.

Topics: Adsorption; Animals; Antifungal Agents; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Defensins; Fungi; Inhibitory Concentration 50; Leukemia; Lymphoma; Mice; Neoplasms; Phaseolus; Phytotherapy; Plant Extracts; Seeds; Sepharose; Triazines

Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC(50) against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Line, Tumor; Circular Dichroism; Erythrocytes; Hemagglutination; Humans; India; Inhibitory Concentration 50; Molecular Sequence Data; Monosaccharides; Neoplasms; Plant Preparations; Protein Binding; Protein Subunits; Ribosome Inactivating Proteins, Type 2; Sepharose; Sequence Homology, Amino Acid; Tandem Mass Spectrometry; Toxins, Biological; Viscum

In magnetic nanoparticle hyperthermia for cancer treatment, controlling the heat distribution and temperature elevations is an immense challenge in clinical applications. In this study we evaluate magnetic nanofluid transport and heat distribution induced by commercially available magnetic nanoparticles injected into the extracellular space of biological tissue using agarose gel with porous structures similar to human tissue. The nanofluid distribution in the gel is examined via digital images of the nanofluid spreading in the gel. A radio-frequency electromagnetic field is applied to the gel following the nanofluid injection and the initial rates of temperature rise at various locations are measured to obtain the specific absorption rate (SAR) distribution. By adjusting the gel concentration and injection flow rate, the results have demonstrated that a relatively low injection rate leads to a spherically shaped nanofluid distribution in the gels which is desirable for controlling temperature elevations. The SAR distribution shows that the nanoparticle distribution in the gel is not uniform with a high concentration of the nanoparticles close to the injection site. We believe that the experimental study is the first step towards providing guidance for designing better treatment protocol for future clinical applications.

Topics: Humans; Magnetics; Nanoparticles; Neoplasms; Sepharose

Radiolabelled DNA-binding compounds can be used to increase the efficiency of radionuclide cancer therapy of disseminated disease. In this work, the aminoacridine compound N-[3-(acridine-9-ylamino)-propyl]-3-iodobenzamide (A3) labelled with the Auger-emitting nuclide 125I using Chloramine-T was studied. Optimal labelling conditions of 125I-A3 were investigated and the interaction with DNA was studied using a novel cell-free in vitro assay with naked human genomic DNA in agarose plugs. This novel assay showed to be simple and reliable. The results verify that 125I-A3 specifically binds DNA with low dissociation and is potent in causing double-strand breaks, yielding 1.0-1.4 breaks per decay. In conclusion, 125I-A3 is a most suitable DNA-binding compound for future therapeutic studies of Auger-electron emitters like 125I.

Topics: Acridines; Benzamides; Biological Assay; Cell-Free System; Chloramines; DNA Adducts; DNA Damage; Humans; Indicators and Reagents; Iodine Radioisotopes; Neoplasms; Sepharose; Tosyl Compounds

Localized activation of the prodrug ifosfamide in or close to tumors by implanting encapsulated ifosfamide-activating cells is an efficacious strategy for tumor therapy. The aim of this study was to evaluate the feasibility of subsieve-size agarose capsules for enclosing the cells in this application. Compared with many conventional microcapsules, subsieve-size agarose capsules are about one-tenth the size and have both higher mechanical stability and allow better molecular exchangeability than other systems. Cells that have been genetically modified to express cytochrome P450 2B1 enzyme were encapsulated in subsieve-size agarose capsules of approximately 90 microm in diameter and implanted into preformed tumors in nude mice. Living cells were detected for >1 month after encapsulation in vitro and showed enzymatic activity (i.e., they were able to activate ifosfamide). More significant regression of preformed tumors was observed in the recipients implanted with cell-enclosing capsules compared with those implanted with empty capsules. These results suggest that the strategy of using subsieve-size agarose capsules enclosing cytochrome P450 2B1-expressing cells is feasible for tumor therapy by chemotherapeutic targeting in combination with ifosfamide administration.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Biophysics; Cats; Cell Line; Cell Size; Cell Survival; Cytochrome P-450 CYP2B1; Drug Delivery Systems; Humans; Ifosfamide; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplasms; Oxazines; Particle Size; Sepharose; Temperature; Time Factors

Isotachophoresis is an electrophoretic method of separation of charged substances. The method is characterized by a discontinuous buffer system, constant velocity of separated molecules, and the distribution of separated components in the form of narrow concentrated bands located one right after another. As a rule, isotachophoresis is not used for the separation of nucleic acids because the mobility of polynucleotides in this system does not depend on their size. However, this circumstance proved to be very useful for the quantitative isolation of heterogeneous DNA fragments from biological fluids, for gene diagnostics of cancer in particular. The proposed method of agarose gel isotachophoresis of DNA has been used for the isolation of blood DNA and its successful PCR analysis.

Topics: Buffers; DNA; DNA, Neoplasm; Electrophoresis, Agar Gel; Genetic Techniques; Humans; Neoplasms; Nucleic Acids; Pancreatic Neoplasms; Polymerase Chain Reaction; Sepharose

The unchecked growth of a solid tumor produces solid stress, causing deformation of the surrounding tissue. This stress can result in clinical complications, especially in confined environments such as the brain, and may also be responsible for pathophysiological anomalies such as the collapse of blood and lymphatic vessels. High stress levels may also inhibit further cell division within tumors. Unfortunately, little is known about the dynamics of stress accumulation in tumors or its effects on cell biology. We present a mathematical model for tumor growth in a confined, elastic environment such as living tissue. The model, developed from theories of thermal expansion using the current configuration of the material element, allows the stresses within the growing tumor and the surrounding medium to be calculated. The experimental observation that confining environments limit the growth of tumor spheroids to less than the limit imposed by nutrient diffusion is incorporated into the model using a stress dependent rate for tumor growth. The model is validated against experiments for MU89 tumor spheroid growth in Type VII agarose gel. Using the mathematical model and the experimental evidence we show that the tumor cell size is reduced by solid stress inside the tumor spheroid. This leads to the interesting possibility that cell size could be a direct indicator of solid stress level inside the tumors in clinical setting.

Topics: Animals; Cell Division; Humans; Models, Biological; Models, Theoretical; Neoplasms; Sepharose; Spheroids, Cellular; Stress, Mechanical; Time Factors

An alpha-Amylase in human liver is detected with an anti-human salivary amylase antibody, but the enzyme activity is very low. We previously found that the rat liver contained an amylase which differed from the enzyme of mice. In this study, we characterized the human liver amylases biochemically and immunohistochermically.. Although the amylase activity of human liver was much lower than that of rat, protein moiety and sugar chains of the human amylase were identified as similar to the rat liver enzyme with an anti-human salivary amylase antibody and by concanavalin A (Con A) affinity chromatography. Liver amylases from human and rat were the same size, 50 kDa, on Western blot analysis and had the same isoelectric points. The cytoplasm of hepatocytes was moderately stained immunohistochemically with the anti-human salivary amylase antibody. Intrahepatic bile ducts were also stained weak-to-moderately. RT-PCR, with a specific primer for the consensus sequence of human amylases, amplified a single 474-bp product from the human liver total RNA. The PCR product was sequenced and referred to the homology. Thirteen bases in the 434-bp fragment of the human liver amylase differed from the corresponding region of the AMY-1 gene transcript and the deduced amino acid sequence differed at five residues. The human liver amylase cDNA sequence was identical to the corresponding cDNA of the AMY-2B, which was known to expressed in tumorous tissues. In situ hybridization revealed the expression of AMY-2B mRNA in non-tumorous human liver.. The present results suggest the possibility that a novel amylase detected in tumorous tissues and encoded by the AMY-2B gene is a liver-specific amylase expressed in the human liver.

Topics: alpha-Amylases; Animals; Base Sequence; Bile Ducts, Intrahepatic; Cytoplasm; DNA, Complementary; Gene Expression; Hepatocytes; Humans; Immunohistochemistry; In Situ Hybridization; Liver; Lung; Molecular Sequence Data; Neoplasms; Pyrimidines; Rats; Reverse Transcriptase Polymerase Chain Reaction; Salivary Glands; Sepharose

To clone full length differentially expressed genes which are related with HBxAg.. HepG2-cells were infected with prepared recombinant retroviruses encoding the X antigen. The differences in gene expression between HepG2 x and HepG2Cat cells were evaluated by suppression subtractive hybridization and PCR. In situ hybridization (ISH) and Northern blot analysis were carried out to screen the differentially expressed genes. The full length cDNA clone of the gene was obtained by 5' and 3' rapid amplification of cDNA ends(race) PCR. HepG2 cells transiently transfected with the new full length gene were subjected to fluorescence activated cell sorting (FACS) analysis for DNA content. HepG2 cells stably transfected with the new full length gene were tested for anchorage independent growth in soft agar and for tumorigenicity in nude mice.. The expression of multiple genes were turned on (8) or off (2) in HepG2X compared to HepG2CAT cells. One differentially expressed gene C2, the human homology of Sui1, encoded a translation initiation factor whose expression was suppressed by X antigen in HepG(2) cells. The full length of this gene was 1.35 kb, which encoded a small protein of 113 amino acids. Introduction of C2 into HepG2 cells could inhibit cell growth in culture, in soft agar, and partially inhibit tumor formation in nude mice. Cells transfected with pcDNA3-HBx showed little or no detectable C2, which was consistent with the suppression of this protein in the presence of HBxAg. C2 was also expressed in nontumor liver, but not in tumor cells from patients with hepatocellular carcinoma.. HBX can regulate the expression of genes whose products may be positive or negative regulators of cell growth. Our work for the first time demonstrates that the mechanism of DNA virus associated carcinogenesis involves altered patterns of gene expression regulated at the level of translation initiation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Culture Media; DNA, Complementary; Eukaryotic Initiation Factor-1; Fungal Proteins; Gene Expression; Humans; Mice; Mice, Nude; Molecular Sequence Data; Neoplasms; Peptide Initiation Factors; Polymerase Chain Reaction; Protein Biosynthesis; Saccharomyces cerevisiae Proteins; Sepharose; Trans-Activators; Tumor Cells, Cultured; Viral Regulatory and Accessory Proteins

We analyse the diffusion problem in the traditional Fe(II/III) agarose gel system employed in MRI studies of radiation dosimetry. The diffusion coefficient is measured using an inversion recovery null-point imaging method in a model gel/water phantom. The diffusion coefficient of Fe(III) in 1% agarose gel at pH 1.1 is D = 2.7 +/- 0.3 x 10(-6) cm2 s-1. The diffusion coefficient of Fe(II) is D = 3.3 +/- 0.5 x 10(-6) cm2 s-1. Measurement of the diffusion coefficients permits simulation of the MRI signal intensity from phantoms with model radiation dose distributions. We allow for diffusion of both Fe(II) and Fe(III) in our simulations as well as the effect of both relaxation agents on the local spin-lattice relaxation time T1. We also analyse the effects of the physical penumbra on the diffusion problem.

Topics: Gels; Humans; Iron; Magnetic Resonance Imaging; Mathematics; Neoplasms; Phantoms, Imaging; Radiotherapy Dosage; Radiotherapy Planning, Computer-Assisted; Sepharose

The success of a predictive assay for radiotherapy relies on the use of one or more tumor cell traits that equate with tumor radioresistance or radiosensitivity. These traits can be divided into intrinsic (genetic) and extrinsic (epi-genetic) factors. Most probably, a tumor's response to radiotherapy will be influenced by both of these sets of traits. Radiobiological analysis of cultured cells derived from explanted tumors of head and neck patients has shown that in vitro survival of tumor cells is not the only factor affecting tumor radiocurability. Two possible reasons are the high degree of selection involved in growing the cells in vitro and the inability to assess the contribution of the cell-cell contact effect with cultured cells. A possible means of overcoming both of these problems would be an assessment of the radiosensitivity of the cell population immediately after removal from the tumor. Since a good correlation exists between intrinsic cellular radioresistance and DNA double-strand break repair (DSBR) as assayed by the Neutral Elution technique [21], we have investigated the feasibility of using asymmetric field inversion gel electrophoresis (AFIGE) in identifying resistant tumor cells in vitro. AFIGE has several advantages over neutral elution in that it is faster (approximately 60-80 samples can be run on the same agarose gel) and, most importantly, one can visualize DNA damage and repair by staining the DNA with ethidium bromide.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carbon Radioisotopes; Carcinoma, Squamous Cell; DNA; DNA Damage; DNA Repair; DNA, Neoplasm; Electrophoresis; Fibroblasts; Humans; Neoplasms; Radiation Tolerance; Sepharose; Thymidine; Tumor Cells, Cultured

The subforms of MM isozyme of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2, CK) in sera obtained from healthy adults and patients were determined by agarose gel isoelectric focusing (IEF). The patients were classified into six groups according to serum CK-MM activities and IEF patterns. The IEF spectra offered useful information on cell hyperplasia, augmented cell membrane permeability, cell destruction and release time of CK-MM in the circulation from the cells for diagnosis, progress observation and prognosis, especially in the cases of chronic hepatic diseases, acute myocardial infarction and muscular dystrophy. Macro CKs were also determined by IEF. Macro CKs could be completely distinguished from each other, and CK isozymes consisting of macro CK type 1 could be presumed by isoelectric points.

Topics: Chromatography, Gel; Creatine Kinase; Health Status; Humans; Immunoenzyme Techniques; Isoelectric Focusing; Isoenzymes; Liver Diseases; Muscular Dystrophies; Myocardial Infarction; Neoplasms; Prognosis; Sepharose

Soluble inhibitors of lymphokine-activated killer (LAK) cell induction were characterized and purified from serous fluids from healthy donors or from patients with advanced cancer. Inhibitory activity in sera from cancer patients was partially absorbed with protein A agarose or anti-human IgG agarose. Following absorption, residual inhibition varied with individual sera, suggesting the presence of IgG-related and non-IgG inhibitory factors, and the proportions varied in the patient population. IgG, purified by affinity chromatography from ascitic fluids, from plasma of cancer patients, and from plasma of healthy donors, significantly inhibited IL-2 induction of LAK cells in a dose-dependent manner. Irrespective of the sources, inhibitory activity resided in the high-molecular-weight IgG fraction composed of IgG aggregates and immune complexes, but not monomeric IgG. Commercially prepared human IgG in aggregated form, but not in monomeric form, inhibited LAK cell induction in a dose-dependent manner. In contrast, neither bovine IgG nor human albumin affected LAK cell induction, even at higher concentrations. Aggregated human IgG inhibited LAK cell induction in unfractionated peripheral blood mononuclear cells (PBMC) but not in monocyte-depleted peripheral blood lymphocytes (PBL). Despite extensive (greater than 99%) depletion of IgG by protein-G affinity chromatography, the serous fluid from cancer patients displayed significant inhibitory activity. Fractionation of the IgG-depleted inhibitory materials by Sephacryl S-300, high-pressure ion-exchange column (HPIEC) or gel-permeation chromatography (HPGPC) demonstrated a 65-kDa inhibitor, distinct from IgG. Affigel-blue affinity chromatography of the 65-kDa fraction depleted albumin but did not remove the inhibitory activity, suggesting that the 65-kDa inhibitor is not serum albumin nor an albumin-bound component. These results suggest that serous fluids from patients with advanced-stage cancer contain 2 distinct regulators for LAK cell induction: (I) aggregated IgG and (2) a 65-kDa inhibitor, distinct from albumin.

Topics: Antibodies, Anti-Idiotypic; Antibody Formation; Blood Proteins; Chromatography, Affinity; Female; Humans; Immunoglobulin G; Killer Cells, Lymphokine-Activated; Leukocytes, Mononuclear; Melanoma; Neoplasms; Nerve Tissue Proteins; Plasma; Sepharose; Staphylococcal Protein A; Suppressor Factors, Immunologic

Plasma from 182 patients with different malignant diseases was tested for riboflavin binding by immunoglobulins, which have been recently identified as major carriers of this micronutrient. A wide range of binding (5.9 to 130 pmole/ml plasma) was observed, and significant elevations were found for patients having breast cancer (21.2 +/- 1.9, P less than 0.05) and melanoma (25.7 +/- 1.9, P less than 0.001) compared to controls (15.5 +/- 1.9). The proteins responsible for a majority of the higher binding were identified as immunoglobulins, based on their elution from gel filtration columns and the removal of 57-88% of the non-albumin binding by treating of plasma with Protein A-agarose. The binding was only weakly related to the total concentration of immunoglobulins (r = 0.11 by linear regression analysis), however, and is apparently due to a subclass that is elevated in some types of cancer. Elevated levels of these immunoglobulins may contribute to the lower urinary levels and clearance of riboflavin in cancer.

Topics: Breast Neoplasms; Chromatography, Gel; Female; Humans; Immunoglobulin G; In Vitro Techniques; Male; Melanoma; Neoplasms; Nephelometry and Turbidimetry; Protein Binding; Riboflavin; Sepharose

Agarose-bound asialofetuin functions as an insoluble sialyl group acceptor in a simplified assay for sialyl transferase (CMP-neuraminate: D-galactosyl-glycoprotein N-acetylneuraminyl transferase; EC 2.4.99.1) in serum. Since sialyl transferase levels in serum are elevated in a large number of malignant conditions, the simplified assay is of use for clinical monitoring in tumour therapeutic programmes.

Topics: alpha-Fetoproteins; Asialoglycoproteins; Clinical Enzyme Tests; Fetuins; Humans; Kinetics; Neoplasms; Reference Values; Sepharose; Sialyltransferases; Transferases

Human nonadherent peripheral blood mononuclear cells (PBMC) isolated from nonimmunized donors were preincubated for 18 h in medium alone or medium containing the lymphokine interleukin 2 and subsequently cocultured with tumor cells derived from malignant tumor cell lines or from fresh human tumors. The cell suspensions were subsequently inoculated into agarose; 14 days later, new tumor colony formation was determined. Although the different tumor cells displayed a wide range of sensitivity to the PBMC, in each instance, the number of colonies formed by the tumor cells exposed to the PBMC was consistently reduced relative to that of control cells. The inhibitory effect on the colony-forming cells was especially pronounced with PBMC preincubated with interleukin-2 and was dependent on the ratio of tumor cells to PBMC in the culture. This assay system provides an alternative to the standard 51Cr release assays in assessing the immunomodulatory effects of lymphokines and in quantitating the cytolytic or cytostatic activity of various effector cells against neoplastic stem cells from established cell lines and from heterogeneous cell preparations derived from fresh human tumors.

Topics: Cell Line; Cytotoxicity, Immunologic; Humans; Interleukin-2; Lymphocyte Activation; Lymphocytes; Neoplasms; Neoplastic Stem Cells; Sepharose; Stem Cells

Specificity of antibodies to Victoria strain of Newcastle disease virus (NDV) found in infectious mononucleosis (IM) and other pathologic sera was investigated by agglutination of NDV-modified human O red blood cells, as well as by immunodiffusion and enzyme immunoassay with various preparations of the virus. These studies clearly demonstrated that the NDV antibodies are distinct from P-B or H-D antibodies. The unexpected observation that guinea pig kidney (GPK) tissues absorbed NDV antibodies allowed their classification into a group of 'GPK-positive' heterophile antibodies. The simultaneous occurrence of the NDV antibodies and H-D antibodies in IM and other diseases suggests the possibility that multiple new antigenic determinants, especially those of carbohydrate nature, may appear due to the alteration of self-antigens as a result of various pathologic processes.

Topics: Antibodies, Viral; Antibody Specificity; Gels; Humans; Immunodiffusion; Infectious Mononucleosis; Leprosy; Lupus Erythematosus, Systemic; Multiple Sclerosis; Neoplasms; Newcastle disease virus; Sepharose; Syphilis

Eleven dogs with spontaneous neoplasms were intensively treated with an immunoadsorption system consisting of a continuous flow centrifuge, Protein A-Sepharose columns, and a semi-automatic elution system. Despite consistent and substantial lowering of immunoglobulin G levels, tumor regression was noted in only one of 11 dogs. In contrast, infusion of small volumes of plasma after incubation with heat and formalin-treated Staphylococcus aureus Cowan I resulted in a tumoricidal response in five of six animals. These results suggest that tumor necrosis is probably not induced by Protein A-mediated removal of humoral "blocking" factors.

Topics: Animals; Cat Diseases; Cats; Dog Diseases; Dogs; Female; Immunoglobulin G; Male; Neoplasms; Perfusion; Plasma; Sepharose; Staphylococcal Protein A; Staphylococcus aureus

Primate neoplastic and finite cell lines were tested in one in vivo and two in vitro test systems: adult nude mice, muscle organ culture (MOC) and soft agarose (SA). Comparison of the sensitivity of the systems indicated that nude mice were inferior to either in vitro system: WI-38 VA13 (an SV40 transformed cell line) did not cause tumours in these animals yet it behaved as if it were neoplastic in MOC and formed colonies in SA. There was complete correlation between results obtained in MOC and SA. All cell lines which produced tumors in vivo were positive in both in vitro test systems. None of the lines which showed normal patterns in MOC and in SA was tumorigenic in nude mice. Since testing in vitro is simpler, faster, and is thought to be reliable, we recommend SA followed by MOC as the initial assays for determining tumorigenicity of cells.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Female; Humans; Mice; Mice, Nude; Muscles; Neoplasm Transplantation; Neoplasms; Organ Culture Techniques; Sepharose

In vitro drug sensitivity testing, both by optical colony counting and by a [3H]-TdR incorporation assay, was performed on human tumour cells proliferating in soft agar cultures. Cells from two different human tumour cell lines, 5 different human tumour xenografts, and 94 different primary human tumour specimens of various histologic types were studied. Regression analysis comparing the results of the colony counting assay and the [3H]-TdR assay revealed good to excellent correlations between the two assay endpoints for quantitating the effect of in vitro anticancer drug exposure for a large number of different agents. The presence of pre-existing tumour cell aggregates complicates the performance of the optical colony counting assay. The [3H]-TdR incorporation assay is more sensitive and reproducible than the colony counting assay when performed on samples containing a large number of initially seeded tumour cell aggregates.

Topics: Antineoplastic Agents; Cell Count; Cell Line; Colony-Forming Units Assay; Female; Humans; Neoplasm Transplantation; Neoplasms; Sepharose; Thymidine; Transplantation, Heterologous; Tritium; Tumor Stem Cell Assay

Two technological problems limit the usefulness of chemosensitivity assays: low success rates (generally 30-60%); and the requirement for large numbers of tumor cells (5 X 10(5)/dish). To solve these problems, we developed a miniaturized, improved, nucleic acid precursor incorporation assay (MINI-assay). In this new assay, 0.3-1.5 X 10(5) tumor cells were plated in double-layer agarose in 16-mm wells of a Costar (No. 3524) 24-well cluster dish. After 72 h of incubation, 5 microCi [3H]thymidine were added to each well. After an additional 24 h of incubation, the trichloroacetic acid-precipitable material was collected and counted by liquid scintillation. We found that 280 of 351 (80%) human solid tumors gave evaluable chemosensitivity results. Labeling efficiency was optimum when the plating density was between 1.5 and 3 X 10(4) cells/well. Radioisotope uptake was less efficient in 35-mm Petri dishes and in the 7-mm wells. The MINI-assay was particularly suitable for small specimens (less than 1 g) and for tumor types that usually yield small numbers of viable tumor cells (19 of 30 breast cancers and 56 of 71 sarcomas were evaluable). The artifacts of colony counting (cell clumps, debris, clots) were also eliminated with this assay. With high evaluability rates, the requirement of fewer cells, a short duration (5 days), and ease of quantitation, the MINI-assay is widely applicable to chemosensitivity testing in human tumors.

Topics: Agar; Cell Count; Colony-Forming Units Assay; Culture Media; Humans; Neoplasms; Sepharose; Thymidine; Tumor Stem Cell Assay

The presence of cellular aggregates in cell suspensions derived from human solid tumours often complicates subsequent evaluation of colony formation in primary soft agar cultures (Agrez et al., 1982b). In the present study, performance of a conventional colony formation assay was observed to lack sufficient sensitivity to identify growth and active chemotherapeutic agents in the majority of specimen cultures. Modification of conventional methodologies to include filtration of cell suspensions, use of "proliferation control" and "cytotoxicity control" cultures as well as vital staining were found to be essential for the valid assessment of primary soft agar cultures in our laboratory. In addition, application of drugs to culture surface in place of culture incorporation appeared to facilitate culture performance and drug sensitivity testing.

Topics: Antineoplastic Agents; Azides; Cells, Cultured; Clone Cells; Colony-Forming Units Assay; Female; Filtration; Humans; Male; Methods; Neoplasms; Sepharose; Sodium Azide; Staining and Labeling; Tumor Stem Cell Assay

Circulating immune complexes (CIC) were determined in tumour patient sera using three methods. One is based on PEG-precipitation, one on C1q-reactivity, and one on protein A-reactivity. About 25-30% of the sera were positive in at least one of the tests. Incubation of serum with protein A-Sepharose in vitro removed PEG-precipitable CIC from most sera, whereas C1q-reactive CICs had a much lower affinity to protein A. The protein A-reactive complexes showed considerable variation in their binding to protein A-Sepharose, and in some sera the amount of these CICs was actually increased. Similar changes in protein A-reactive CIC were also found during ex vivo treatment of tumour patients with immune adsorption. It is proposed that the binding of immune complexes to protein A can result in remodelling of protein A itself. Results from ultracentrifugation and fractionated PEG-precipitation support this hypothesis.

Topics: Antigen-Antibody Complex; Blood; Chromatography, Affinity; Complement Activating Enzymes; Complement C1q; Fractional Precipitation; Humans; Immunosorbent Techniques; Neoplasms; Plasmapheresis; Sepharose; Staphylococcal Protein A

Protein A (SpA) alone or immobilized on bacteria (e.g., Cowan strain I), collodion charcoal, or on Sepharose have been used in serotherapy of cancer in humans and experimental animals. Because SpA forms complexes with IgG that can activate complement, and the physiologic response during treatment often involves hypocomplementemia and reactions that are similar to those induced by anaphylatoxins, we used sensitive and specific radioimmunoassays to test the ability of SpA reagents to generate C3a, C4a, and C5a from human serum. The yield of anaphylatoxins depended on the dose of SpA, with the maximum generation of C3a (47 to 55 micrograms/ml) and C5a (1.4 to 1.9 micrograms/ml) being produced with levels of SpA that were maximally precipitated from serum. Maximum C4a levels (up to 15 micrograms/ml) were obtained at concentrations of SpA equal to or greater than the dose required to give optimal precipitation. The maximum concentrations of anaphylatoxins correspond to essentially quantitative conversions of C3 to C3a, C4 to C4a, and 40% of C5 to C5a after correction for levels found in serum incubated in pyrogen-free saline. Preformed insoluble complexes prepared from either serum or monomeric IgG also were capable of generating anaphylatoxins in fresh whole serum up to levels approximately equal to those observed in serum treated directly with an optimal amount of SpA. The preformed complexes from serum or IgG generated similar high concentrations of anaphylatoxins when carried through four sequential incubations with fresh serum, and complexes that contained approximately 1 microgram SpA were still active. Preincubating the insoluble complexes with chicken anti-SpA serum did not alter their activity. Incubation of serum with collodion charcoal coated with SpA, in a system that models the perfusion technique used to treat cancer, produced complexes that generated significant levels of C3a compared with levels found in serum passaged over albumin charcoal or in untreated serum. The C3a levels in serum from the albumin collodion charcoal were not significantly different from those found in untreated serum. Similar amounts of C3a, C4a, or C5a were observed in serum incubated with differing numbers of bacteria representing a strain of S. aureus rich in cell bound SpA (Cowan strain I) or a strain (Wood 46) deficient in SpA. This suggests that in intact bacteria, cell wall factors other than SpA (e.g., peptidoglycan) are predominantly responsible for generating an

Topics: Anaphylatoxins; Animals; Charcoal; Complement C3; Complement C3a; Complement C4; Complement C4a; Complement C5; Complement C5a; Humans; Immunization, Passive; Neoplasms; Peptide Biosynthesis; Rabbits; Sepharose; Staphylococcal Protein A; Staphylococcus aureus

A specific and sensitive enzyme immunoassay system for human secretory IgA was developed using anti-alpha-chain antibodies coupled to activated thiol-Sepharose, and anti-secretory component antibodies labeled with beta-D-galactosidase from Escherichia coli. The dose response of the enzyme activity in eluate was observed between 3 and 1000 ng of secretory IgA with little cross-reactions with IgA, IgG, IgM and secretory component. The assay method could be employed for the measurement of secretory IgA in saliva, urine, feces, intestine and serum without interferences by the abundant IgA in the same samples.

Topics: Animals; Child, Preschool; Cross Reactions; Feces; Humans; Immunoenzyme Techniques; Immunoglobulin A; Immunoglobulin A, Secretory; Infant; Infant, Newborn; Intestines; Neoplasms; Polysaccharides; Rabbits; Saliva; Sepharose; Sulfhydryl Compounds; Urine

Human tumor cell lines, derived from cancers of the colon, ovary, and cervix, were grown in liquid tissue culture media and media made semisolid with agar (Bacto + deoxycholate lactose agar), agarose [LE, ME, Sea Plaque and Sea Prep (15/45)], and methyl cellulose. The effects of each agent on overall cell proliferation and rate of overall cell proliferation were examined. The agents, used to make media semisolid, were observed to inhibit or, in some cases, enhance cell growth in a fashion that was characteristic of individual cell lines. These phenomena may be of consequence to the optimization of nutrient media for primary tumor cell preparations.

Topics: Agar; Cell Division; Cells, Cultured; Culture Media; Humans; Methylcellulose; Neoplasms; Sepharose

Mason-Pfizer monkey virus (MPMV) and PMFV, an isolate from a human continuous cell line, were compared by Sepharose bead immunofluorescence assay. According to the results with p27-specific assays the main structural protein of both viruses seems to be identical in the prominent antigenic determinants. Differences were found when comparing the p15s indicating that this viral protein contains type-specific antigenic determinants.

Topics: Animals; Antigens, Viral; Cell Line; Dogs; Electrophoresis, Polyacrylamide Gel; Epitopes; Female; Fluorescent Antibody Technique; Humans; Lung; Macaca mulatta; Neoplasms; Retroviridae; Sepharose; Thymus Gland; Viral Proteins

A considerably simplified assay for recording sialyl- and fucosyltransferase in human serum is presented. Serum samples incubated with labeled nucleotide-sugar and glycosylated endogenous acceptor molecules were adsorbed to Con A Sepharose and quantitated by scintillation counting. The results correlated with those of a much more time consuming acid precipition method, and displayed a higher diagnostic sensitivity due to the improved specificity of the method and the combined recording of the two activities. A correlation between serum sialyl- and fucosyltransferase activities as well as quantitative agreement between the amount of incorporated sialic acid and fucose indicated that rhe endogenous acceptor molecules were rate-limiting for transfer and may themselves have diagnostic potential.

Topics: Adsorption; Clinical Enzyme Tests; Fucosyltransferases; Hexosyltransferases; Humans; Methods; Neoplasms; Polysaccharides; Sepharose; Sialyltransferases; Transferases

Leukocyte migration tests under agarose (Clausen technique) were performed in 28 patients tentatively diagnosed as having any malignancy with the use of a 3 M KCl-extract panel prepared from bronchogenic, gastric, colonic, renal, and mammary carcinoma, corresponding normal tissues, carcinoembryonic antigen (CEA), and human encephalitogenic protein (HEP). 17 out of 22 proven carcinoma patients showed sensitization by reaction with optimal concentrated KCl-extract of cancer from the same organ type as their own tumor. In some cases positive reactions could be observed also with normal tissue antigen (NTA) of tumor organ type (7/22) or with an additional carcinoma extract of organ type differing from patients own primary tumor (8/22). Gastrointestinal carcinomas, especially, showed sensitization to CEA (7/12) contrary to nongastrointestinal carcinomas (1/10). With HEP no positive reactivity could be found (0/10). With the use of tumor antigen panel (5 antigens) only few positive reactions (MI less than 0.80 or greater than 1.20) could be observed in 6 patients with nonmalignant diseases (1/30 tests) and 8 healthy blood donors (1/40 tests). A widespread individual screening program using tissue antigens for patients suspected of malignancies could give a pattern of reactivities and improve the recognition of cell-mediated sensitization against tumor tissues.

Topics: Aged; Antigens, Neoplasm; Breast Neoplasms; Carcinoembryonic Antigen; Cell Migration Inhibition; Colonic Neoplasms; Epitopes; Female; Humans; Immunity, Cellular; Kidney Neoplasms; Leukocytes; Lung Neoplasms; Male; Middle Aged; Myelin Basic Protein; Neoplasms; Sepharose; Stomach Neoplasms

Topics: Acid Phosphatase; Antibody Specificity; Fluorescent Antibody Technique; Humans; Immunoglobulin G; Male; Neoplasms; Prostatic Neoplasms; Sepharose

Topics: BCG Vaccine; Cell Migration Inhibition; Humans; Hypersensitivity, Delayed; In Vitro Techniques; Leukocytes; Melanoma; Neoplasms; Sepharose; Tuberculin; Tuberculin Test

Topics: Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Autoantibodies; Epitopes; Fluorescent Antibody Technique; Humans; Neoplasms; Ovalbumin; Rats; Sepharose

The defined antigen substrate spheres (DASS) system was employed for analysing reactions between solid state antibody or antigen and soluble immune complexes. Sepharose beads covalently coupled with ovalbumin were used to represent tumour cells and beads coupled with antibody against ovalbumin were used to represent anti-tumour lymphocytes; the ovalbumin and corresponding antibody simulated tumour-derived antigen and antibody to tumour respectively. Binding of soluble complexes to the beads was measured by fluorimetry and/or radiometry of fluorescein or 125-I-labelled ovalbumin or antibody. Antigen-antibody complexes in antibody excess bound less effectively to the antibody beads than antigen alone, but complexes in slight or moderate antigen excess bound more effectively. Complexes in antibody excess were most effective in the complex before levelling off and then decreased in extreme antibody excess. The model demonstration of augmentation by antibody of antigen binding to solid state antibody might by analogy reflect a mechanism of inhibition of lymphocyte cytotoxicity. Complexes in a wide range of antibody excess should also be effective in blocking lymphocytotoxicity at the target cell level.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Antigen-Antibody Reactions; Antigens; Antigens, Neoplasm; Fluorescent Antibody Technique; Guinea Pigs; Lymphocytes; Models, Biological; Neoplasms; Ovalbumin; Sepharose