Biospecific adsorption in fixed and periodic countercurrent beds

A mathematical model that describes the adsorption and wash stages of biospecific adsorption (affinity chromatography) in a packed column is presented. The model expressions account for film and pre diffusion mass transfer as well as for different mechanisms of interaction between the adsorbate(s) and the ligand. The model equations may be applicable to single and multi-component biospecific adsorption systems involving both monovalent and multivalent adsorbates.The results obtained from model simulations show that the breakthrough time of the adsorbate is significantly influenced by the rate of the interaction step between the adsorbate and the ligand. The results indicate that when short beds are employed, then the choice of ligand with respect to its rate of interaction with the adsorbate may be of paramount importance. In certain systems involving bivalent adsorbates, the adsorbate may be displaced from the one-site complex, reenter the flowing fluid stream, and increase the effluent adsorbate concentration above its inlet value. It is also shown that when a single column is divided into two beds operating in a periodic counter current mode, the ligand utilization can be almost four times higher than that obtained in a column of the same length operating in the fixed bed mode.The studies on the wash stage indicate that the reduction of the concentration of the contaminant to a specified low level may be accomplished for certain systems in a shorter time, if the direction of flow in the wash stage is opposite to that used in the adsorption stage. However, a larger amount of product will be lost, in general, when the direction of flow of the washing medium is opposite to that employed during the adsorption stage.     

Modeling of the primary and secondary drying stages of the freeze drying of pharmaceutical products in vials: numerical results obtained from the solution of a dynamic and spatially multi-dimensional lyophilization model for different operational policies

A rigorous unsteady state and spatially multidimensional model is presented and solved to describe the dynamic behavior of the primary and secondary drying stages of the lyophilization of a pharmaceutical product in vials for different operational policies. The results in this work strongly motivate the aggressive control of freeze drying and it is found that heat input control that runs the process close to the melting and scorch temperature constraints yields (i) faster drying times, and (ii) more uniform distributions of temperature and concentration of bound water at the end of the secondary drying stage.     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

Pore network modelling of affinity chromatography: determination of the dynamic profiles of the pore diffusivity of beta-galactosidase and its effect on column performance as the loading of beta-galactosidase onto anti-beta-galactosidase varies with time.

A three-dimensional pore network model for diffusion in porous adsorbent particles was employed in a dynamic adsorption model that simulates the adsorption of a solute in porous particles packed in a chromatographic column. The solution of the combined model yielded the dynamic profiles of the pore diffusion coefficient of beta-galactosidase along the radius of porous adsorbent particles and along the length of the column as the loading of beta-galactosidase onto anti-beta-galactosidase immobilized on the surface of the pores of the particles occurred, and, the dynamic adsorptive capacity of the chromatographic column as a function of the design and operational parameters of the chromatographic system. It was found that for a given column length the dynamic profiles of the pore diffusion coefficient were influenced by (a) the superficial fluid velocity in the column, (b) the diameter of the adsorbent particles, and (c) the pore connectivity of the porous structure of the adsorbent particles. The effect of the magnitude of the pore connectivity on the dynamic profiles of the pore diffusion coefficient of beta-galactosidase increased as the diameter of the adsorbent particles and the superficial fluid velocity in the column increased. The dynamic adsorptive capacity of the column increased as (i) the particle diameter and the superficial fluid velocity in the column decreased, and (ii) the column length and the pore connectivity increased. In preparative affinity chromatography, it is desirable to obtain high throughputs within acceptable pressure gradients, and this may require the employment of larger diameter adsorbent particles. In such a case, longer column lengths satisfying acceptable pressure gradients with adsorbent particles having higher pore connectivity values could provide high dynamic adsorptive capacities. An alternative chromatographic system could be comprised of a long column packed with large particles which have fractal pores (fractal particles) that have high pore connectivities and which allow high intraparticle diffusional and convective flow mass transfer rates providing high throughputs and high dynamic adsorptive capacities. If large scale monoliths could be made to be reproducible and operationally stable, they could also offer an alternative mode of operation that could provide high throughputs and high dynamic adsorptive capacities.     

High affinity glycosaminoglycan and autoantigen interaction explains joint specificity in a mouse model of rheumatoid arthritis

In the K/BxN mouse model of rheumatoid arthritis, autoantibodies specific for glucose-6-phosphate isomerase (GPI) can transfer joint-specific inflammation to most strains of normal mice. Binding of GPI and autoantibody to the joint surface is a prerequisite for joint-specific inflammation. However, how GPI localizes to the joint remains unclear. We show that glycosaminoglycans (GAGs) are the high affinity (83 nm) joint receptors for GPI. The binding affinity and structural differences between mouse paw/ankle GAGs and elbows/knee GAGs correlated with the distal to proximal disease severity in these joints. We found that cartilage surface GPI binding was greatly reduced by either chondroitinase ABC or beta-glucuronidase treatment. We also identified several inhibitors that inhibit both GPI/GAG interaction and GPI enzymatic activities, which suggests that the GPI GAG-binding domain overlaps with the active site of GPI enzyme. Our studies raise the possibility that GAGs are the receptors for other autoantigens involved in joint-specific inflammatory responses.     

Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells

The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.     

Midline signaling regulates kidney positioning but not nephrogenesis through Shh

The role of axial structures, especially the notochord, in metanephric kidney development has not been directly examined. Here, we showed that disruption of the notochord and floor plate by diphtheria toxin (DTA)-mediated cell ablation did not disrupt nephrogenesis, but resulted in kidney fusions, resembling horseshoe kidneys in humans. Axial disruptions led to more medially positioned metanephric mesenchyme (MM) in midgestation. However, neither axial disruption nor the ensuing positional shift of the MM affected the formation of nephrons and other structures within the kidney. Response to Shh signaling was greatly reduced in midline cell populations in the mutants. To further ascertain the molecular mechanism underlying these abnormalities, we specifically inactivated Shh in the notochord and floor plate. We found that depleting the axial source of Shh was sufficient to cause kidney fusion, even in the presence of the notochord. These results suggested that the notochord is dispensable for nephrogenesis but required for the correct positioning of the metanephric kidney. Axial Shh signal appears to be critical in conferring the effects of axial structures on kidney positioning along the mediolateral axis. These studies also provide insights into the pathogenesis of horseshoe kidneys and how congenital kidney defects can be caused by signals outside the renal primordia.