David M. Yarmush

An analysis of transport resistances in the operation of BIAcore; implications for kinetic studies of biospecific interactions.

The mass-transfer processes that affect kinetic measurements of biospecific interactions between one species in a flowing solution and another species immobilized in a thin hydrogel instrument were analysed by means of convection-diffusion reaction models. The specific purpose was to identify experimental design considerations for kinetic measurements using the BIAcore instrument. Numerical solutions identified three different regimes of operation. A kinetic regime exists at low values of Damkohler number (Da), when the intrinsic kinetics are slow and the diffusion is relatively fast. This allows for the accurate determination of kinetic constants. A limiting value of Da, above which mass-transfer limitations appear, is presented as a function of Peclet number, Pe. At higher Da values, the reaction occurs in the mass-transfer-controlled regime where the reaction-rate is independent of the intrinsic kinetics. It was observed that, frequently, the reaction occured in an intermediate regime where, although the mass-transfer rate was not strictly limiting, substantial concentration gradients were present. Analysing the data in this regime by direct application of kinetic equations underestimates the association rate constant. Even when the reaction was not limited by mass-transfer in the flow channel, it may have been affected by steric hindrance to transport in the hydrogel, if a large concentration of capturing antibody or ligand was immobilized. The primary effect of the hindrance was to lower the soluble-species (analyte) concentration in the hydrogel when compared to the bulk solution. Non-uniformity of conditions within the hydrogel in the presence of steric hindrance had a significant effect on the observed reaction. The effect was most prominent at higher analyte concentration, when the rate constant showed an apparent reduction as the reaction progressed.

Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis

mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3-8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography-tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPbeta6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.

Monoclonal antibody-chromophore conjugates as selective phototoxins

Abstract he chromophore chlorin e 6 monoethylene diamine monoamide (CMA) was bound via poly(glutamic acid) (PGA) to two monoclonal antibodies (Mabs), an anti bovine serum albumin (BSA) Mab designated 9.1 and an anti-Leu-1 Mab. The final ratio of chromophore to Mab was dependent on the starting concentration of the chromophore. Binding activity and specificity, determined using 125 I-labeled Mab, showed 70% retention at CMA:Mab ratios of 36. Singlet oxygen quantum yields relative to rose bengal, estimated from the bleaching of N , N ->dimethylnitrosoaniline, were the same for CMA free or bound to Mab. When Mab 9.1 was conjugated to a diamino cyanine, 1,1′-(3-propylamine hydrobromide)-3,3,3′,3′-tetramethylindotricarbocyanine (TCC), the resultant conjugates were highly aggregated, presumably due to crosslinking via the two amino groups. When anti-Leu-1 target cells, HPB-ALL T-leukemia cells treated with the conjugate were exposed to broad-band, long wavelength irradiation and cell survival evaluated by fluorescence microscopy, a radiant-exposure-dependent phototoxicity was observed. Cells treated similarly with chlorin e 6 , buffer or Mab alone were unaffected. At the concentrations used in these experiments, neither CMA nor anti-Leu-1 were toxic in the absence of light. These results suggest that conjugates having high chromophore to Mab ratios can be prepared using PGA, and that these conjugates may find use as potential photochemotherapeutic agents.

Immunoaffinity purification: Basic principles and operational considerations

Immunoaffinity purification has become an important technique in biotechnology. In this review the basic principles of immunoaffinity separations are described with respect to the stages of operation and potential application. The most commonly used support materials, activation procedures, and coupling chemistries are compared to one another for suitability in various applications. Individual operational steps for fixed bed immunoadsorbents including loading, washing, elution and regeneration are described in terms of both theory and practice. Factors influencing adsorbent stability are identified, and alternative operation and configuration strategies are discussed in light of their application to immunoaffinity systems.

Cutaneous burn injury alters relative tricarboxylic acid cycle fluxes in rat liver.

Severe injury induces a hypermetabolic state in the liver; however, the pathways that are responsible for the increase in hepatic energy demand have not been identified. Relative fluxes in the tricarboxylic acid (TCA) cycle were determined in perfused livers from rats 4 days after administration of a cutaneous burn injury. The perfusate was supplemented with 5 mM uniformly labeled 13C-lactate to efficiently label intracellular metabolites. Flux ratios were calculated on the basis of (1) the 13C-labeling pattern of the glutamate and lactate isotopomers within the liver as determined by nuclear magnetic resonance spectroscopy and (2) an isotopomer mass balance model of the TCA cycle. Calculated flux ratios suggest that burn injury results in an increase in the contribution of pyruvate to the oxaloacetate pool at the expense of non-TCA cycle sources. Furthermore, a dramatic increase in 13C-labeling of glucose was observed in burned rat livers. These data taken together suggest that burn injury induces intrinsic changes in intrahepatic metabolism, including an alteration of the relative fluxes consistent with increased gluconeogenesis from lactate.

Monoclonal antibodies to bovine serum albumin: Affinity and specificity determinations

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.

Quasi-elastic light scattering of antigen-antibody complexes☆

Many biological properties of immune complexes (IC) depend upon their size. Quasi-elastic light scattering (QLS) was used to measure a mean equivalent hydrodynamic radius (Rh) and variance of the distribution of model IC composed of bovine serum albumin (BSA) as antigen (Ag) and combinations of two or three well-characterized monoclonal antibodies (MAb) which bound noncompetitively to unique epitopes on BSA. With the molar ratio (X) of each MAb to Ag fixed, Rh increased with concn. Rh was maximal at equivalence (X = 0.5) with two MAb and at slight MAb excess (X = 0.67) with three MAb. The largest Rh with two MAb was about 200 A, and Rh was uniformly different amongst the three combinations of two MAb IC. The largest hydrodynamic radius of individual complexes which formed with two MAb was estimated to be about 400 A; even larger individual complexes were formed with three MAb. Size changes following alteration of solution concns were also followed with QLS. Kinetics of two MAb IC association were too fast to observe; dissociation following large dilution (40-fold) required 5-10 min to attain a new steady state, much less at small dilution. With three MAb, Rh dropped sharply in 5 min and became steady after 1-2 hr. These results suggest that conventional chromatographic and ultracentrifugation techniques for studying IC size, involving large dilution and long measurement time, provide misleading results. Association of three MAb produced a rapid initial increase of Rh in several min, followed by diverse behavior which depended upon concn. From high to low concn, these included (1) exponential growth of Rh with time and appearance of visible macroscopic particles; (2) metastable states for several hr followed by slow growth to large size over several days, leading to formation of particles; and (3) rapid growth to steady state conditions with no visible particles. This heretofore unobserved equilibrium and kinetic behavior of model IC in solution may be reflected in the behavior of more complex, naturally occurring IC.

A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy

Quasi-elastic light scattering (QLS) was used to determine relative epitope specificities of a group of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb). QLS is a non-invasive technique which can determine the mean size and size distributions of macromolecular scatterers by analysis of the fluctuations in the intensity of laser light scattering. When two MAbs are mixed together with antigen, QLS detects the complex formation which results from the Ag-Ab reaction, and can easily distinguish between the large complexes formed by interaction of non-competitive MAbs and the smaller complexes formed by competitive MAbs. In this report, the competitive or non-competitive behavior of six anti-BSA MAbs were assessed by radioimmunoassay (RIA) and QLS analysis. The results obtained by QLS analysis confirmed the RIA findings indicating that the six MAbs examined can be categorized into three distinct, non-interacting groups. QLS analysis represents a simple, and extremely rapid technique for epitope mapping studies.

Antibody-targeted Photolysis

The technique of Antibody Targeted Photolysis (ATPL) is reviewed from an historical perspective with a summary of the literature since the first experiments were performed in 1983. Attention is given to both the biological and photophysical properties of the various immunoconjugates that have been developed. References to critical discoveries and competing technologies in the photodynamic literature are given. Topics include: synthesis of immunoconjugates, in vitro vs. in vivo toxicity, in vivo biodistribution, immunoconjugate delivery, photosensitizer selection based on photophysical properties, light delivery for specific applications, oxygen requirements, and other physicochemical phenomena. A mathematical model of the dynamics for cell killing based upon the transport of phototoxins to the cell surface is developed. A generalized set of coupled differential equations is given, which conveniently summarizes the manifold requirements stressed earlier for successful cell killing. Solutions are then presented for an idealized set of conditions appropriate for an isolated tumor cell. Suggestions for further improvements and follow-up experiments are made that could help in the evolution of ATPL into a useful clinical therapy and/or probe for cell biological studies.

Burn-induced immunosuppression: attenuated T cell signaling independent of IFN-γ- and nitric oxide-mediated pathways

Burn injury results in immunosuppression; previous work implicated a combination of altered T lymphocyte subpopulations and the elaboration of macrophage‐derived mediators. However, the conclusions were based on T cell stimulations in the setting of high‐dose polyclonal mitogenic stimuli and a single kinetic time‐point. In this study, splenocytes from burned animals were used to examine lymphocyte responses over a multi‐day time course following saturating and subsaturating anti‐CD3, as well as mixed lymphocyte response (MLR) stimulation. Burn injury resulted in suppressed splenocyte‐proliferative responses to high‐dose anti‐CD3 (2 μg/ml) at all culture time‐points (Days 2–5); this inhibition was eliminated by removing macrophages from the splenocyte cultures, by blocking NO production, or by using splenocytes from burned animals congenitally deficient in IFN‐γ (IFN‐γ−/−). The results are consistent with immunosuppression attributable to burn‐induced IFN‐γ production, which in turn, drives macrophage NO synthesis (NOS). In MLR cultures, lymphocyte proliferation and IFN‐γ production were depressed at later time‐points (Days 3–5). APC from burned animals showed no defects as MLR stimulators; T cells from burned animals showed defective, proliferative responses, regardless of the stimulator population. Removing macrophages, adding a NOS inhibitor, or using IFN‐γ−/− splenocytes did not restore the MLR response of burned splenocytes. T cells from burned IFN‐γ−/− animals also showed depressed proliferation with subsaturating levels of anti‐CD3 (0.1 μg/ml); anti‐CD‐28 augmented the proliferative response. We conclude that burn‐induced immunosuppression to authentic antigenic stimulation is related at least in part to defective CD3 signaling pathways and not simply to increased IFN‐γ or NO production.