Hans von Baeyer

Surface reactions on blood contact during haemodialysis and haemofiltration with various membrane types

Abstract The morphological results of blood-surface interaction with artificial membranes utilized in renal replacement devices were investigated. Membrane specimens were obtained from cuprophan, cellulose diacetate, symmetric and asymmetric polyacrylonitrile, polyamide, polysulfone and poly(methyl methacrylate). The membranes were examined by scanning and transmission electron microscopy in their native state and after application. The findings indicate three classes of morphologic blood-membrane interactions: Symmetric type: continuous fibrin precipitation including white blood cells (cuprophan, cellulose diacetate, symmetric polyacrylonitrile); asymmetric type: surface remains free except for a few white blood cells and platelets occasionally attached (polysulfone, asymmetric polyacrylonitrile, polyamide); glass type: surface is covered with a mural white thrombus (poly(methyl methacrylate)). These phenomena can be explained hypothetically by an extension of the “surface mobility hypothesis” of Merrill. The more rigid surface of symmetric membranes hinders sterically the action of heparin near the membrane wall. Microelasticity of asymmetric membranes avoids such inhibition.

Cascade plasmapheresis with online membrane regeneration: Laboratory and clinical studies

Abstract The function of a protein permeable membrane (cellulose diacetate) in a hollow-fiber module was investigated with regard to its application in therapeutic plasmapheresis. The membrane serves as a secondary system element and filters physiological plasma proteins, retaining pathogenic proteins located at the heavy end of the plasma protein spectrum. Membrane fouling, resulting from deposition and immobilisation of some types of protein, is remedied by dilution of the plasma (1:12) and repeated back-flushing of the membrane. The membrane under investigation is anisotropic, and conventional filtration (inside → outside) differs from reverse filtration (outside → inside) both in filtration dynamics and protein sieving. Sieving coefficients for a given protein are substantially lower in whole plasma than in a monospecies solution. This holds for both filtration modes. Therefore, the concept of a “hybrid membrane” [7,8], which implies that both the structure of the membrane and protein—membrane interaction determine functional performance, would seem to be appropriate. Use of the membrane and filtration system in plasmapheresis treatment of patients with familial hypercholesterolemia is described. Retrieval fractions for albumin and IgG, the most important plasma proteins with long effective half-lives, are 50-60% of those with conventional filtration using the XK-60 module (Asahi Medical, Japan)

Selective endocytosis of fluorothymidine and azidothymidine coupled to LDL into HIV infected mononuclear cells

Drug targeting via lipoproteins may be of benefit for use of cytotoxic drugs like fluorothymidine (FLT) or azidothymidine (AZT). Both drugs are potent inhibitors of the human immunodeficiency virus (HIV) reverse transcriptase and are used in the therapy of HIV infection. With regard to this project, the selective endocytosis in HIV infected human macrophages was studied after covalent coupling of AZT and LDL to low density lipoproteins (LDL). Cultured human macrophages and the lymphocytic Molt 4/8 cell line were infected with HIV-1 in vitro and subsequently treated with FLT-LDL or AZT-LDL. Viral replication was followed by determination of cell-released capsid antigen p24. Internalisation into HIV-1 infected human macrophages by the scavenger receptor pathway leads to a dose dependent inhibition of HIV replication. Otherwise, in HIV infected, but scavenger receptor missing lymphocytes (Molt 4/8 cells), neither endocytosis nor inhibition of HIV replication results. Thus, covalent coupling of drugs to LDL leads to a macrophage specific transport. This strategy could possibly avoid toxic side effects in the therapeutic use of antiretroviral drugs and thus may open a way for an earlier chemotherapy in HIV infection.

Preparation of Nucleoside-LDL-Conjugates for the Study of Cell-Selective Internalization: Stability Characteristics and Receptor Affinity

Antiviral therapy of human immunodeficiency virus (HIV) infection is currently based on inhibition of reverse transcriptase by dideoxynucleosides, such as azidothymidine. Because of widespread toxicity it is reasonable to selectively target these drugs to infected cells. This may be accomplished utilizing drug-LDL conjugates, which are internalized via cell specific receptor pathways. With respect to HIV infection, scavenger receptors of the macrophage system seems to offer a hopeful perspective. This pathway requires chemical modification of surface polarity of the LDL. Cell experiments were conducted in HepG2 hepatocytes, which express apolipoprotein B receptors, and in P388 macrophages, which express scavenger receptors. LDL particles to be conjugated were isolated from blood donor plasma and from LDL-apheresis waste material. Non-covalent LDL conjugation with amphiphilic nucleoside derivatives produced only an unspecific nucleoside transfer to cell membranes, due to instability of the LDL conjugates. An experimental method (coincubation test) was developed to identify those conjugates that are stable in the presence of other lipophilic compartments. Covalent coupling of nucleosides to the apolipoprotein B moiety of LDL particles resulted in stable conjugates. As a consequence, the surface charge became negative, and the LDL displayed scavenger receptor affinity rather than apolipoprotein B receptor affinity. Selective targeting of nucleosides to macrophages can be accomplished by covalent coupling to LDL.

Membrane plasma protein fractionation for low-density lipoprotein apheresis: comparison of two hollow fiber modules and two filtration systems

Abstract Human plasma was separated by plasmapheresis from patients with familial hypercholesterolemia and fractionated using two different hollow fiber membrane modules and two different filtration techniques. The goal of the membrane plasma protein fractionation process was to eliminate excess low-density lipoproteins (LDL) from the separated plasma and to return the LDL-purified plasma (with all other physiological proteins) to the patient. The major obstacles encountered in the filtration of macromolecular solutions are concentration polarization, membrane fouling and pore plugging. The loss of inherent membrane characteristics due to these secondary filtration effects results in a drastically reduced filtration flux and decreased protein sieving. This study shows that the desired fractionation into graded protein size classes can be accomplished when appropriate membranes and controlled filtration conditions are used. In a total of 273 LDL apheresis treatments in 9 patients, the improved filtration technique, the FU system, demonstrates that ultrafiltration is a suitable fractionation process and therapeutic substitution-free LDL plasmapheresis is possible.

Permanent-use haemofilter based on porous glass capillaries

Modules of surface-modified porous glass capillaries with a hydraulic conductivity of 0.25-0.70 ml min-1 mmHg-1 m-2 were tested for suitability for clinical haemofiltration by ex vivo dog experiments and by in vitro perfusion. The results show that (1) the construction of reusable haemofilters based on porous glass capillaries is possible; (2) operational data of testmodules are comparable with hollow-fiber high-flux haemofilters based on asymmetric cellulose acetate membranes; and (3) blood-surface interaction of porous glass capillaries is characterized by protein deposition which entails very low protein cutoff.

Fundamentals and application of differential filtration

The fractionation of macrosolute mixtures into classes of graded molecular sizes is the most difficult and, at the same time, most desirable membrane moderated process, according to W.F. Blatt. The major problems originate from concentration polarization, pore plugging, and membrane fouling. Up until now there has been no comprehensive theory that describes these filtration effects satisfactorily. Thus, all accomplishments of differential filtration techniques are based mainly on empirical knowledge. According to concentration polarization phenomena, increasing membrane wall concentrations of rejected macromolecules superimpose an additional membrane resistance. This dynamically formed “hybrid membrane” may govern the filtration process, exerting a dual effect upon hydraulic and solute permeability. Experience obtained by studying the physics of differential filtration guided us to develop a system using currently available artificial membranes for clinical application. This system uses membrane modules with a nominal cutoff of 0.6 mega Dalton, which enables the separation of macroproteins out of the spectrum of plasma proteins. Thus, this system can be applied for the hemapheretic treatment of hypercholesterolemia with excessively high levels of low density lipoprotein (LDL). This overview contains some basic principles of differential filtration and the data of its clinical use. Clinical observations as to regression of atherosclerotic lesions are not included, but are intended for separate publication.