C. William Castor

An improved method for immobilizing IgG antibodies on protein A-agarose

This report describes a modification of a procedure developed by others for crosslinking IgG to protein A which itself is covalently linked to a gel support. Earlier immunoaffinity columns were described as having large antigen-binding capacities and stability under a variety of elution conditions. The present data show that columns constructed with earlier techniques were only partially stable to pH 3.0 buffers, and, as a result, bound less than 20% of the antigen predicted by theory. Modifying parameters of the dimethylpimelimidate crosslinking method led to immunoaffinity columns which did not leak immunoglobulin under low pH elution buffer conditions. The new immunoaffinity absorbants, because of the increased strength of the couple between the antibody and protein A, were capable of binding antigen at over 80% of their theoretical capacity.

SYNOVIAL CELL ACTIVATION INDUCED BY A POLYPEPTIDE MEDIATOR

Rheumatoid arthritis is exclusively a disease of man, and lack of an experimental animal model has impeded progress in understanding the etiology and pathogenesis of this destructive process. At the level of the synovial membrane, the rheumatoid process is characterized by intermittent connective tissue cell proliferation, overproduction of underpolymerized hyaluronic acid,’ increased glycolysis,2 colonization by various types of inflammatory cells, and abnormalities of the microvasculature.3 In the hope of developing a relevant in vitro investigative model, this laboratory established 61 synovial cell strains from normal individuals and patients with different forms of arthritis. The “abnormalities” detected in the rheumatoid cell strains (TABLE 1) were of particular interest, because these characteristics were propagated from one generation of cells to the Efforts to reproduce the “rheumatoid” characteristics in normal synovial cells by adding rheumatoid sera to the media lead to minor and inconsistent alterations in cellular behavior.‘j Because evidence for humoral factors capable of inducing “rheumatoid behavior” in normal synovial cells was weak, we next examined the response of normal synovial cells to selected cellular factors. Isolated human peripheral blood lymphocytes, polymorphonuclear leukocytes, and platelets were cocultured with monolayer cultures of normal human synovial cells and found to cause profound changes in culture activity. These changes included decreased medium pH, marked acceleration of hyaluronic acid synthesis, and striking increases in glucose uptake and lactic acid formation.’ It soon became clear that slurries of dead leukocytes (frozen-thawed) elicited the same hypermetabolic synovial cell response. Extracts of both syngeneic and allogeneic leukocytes stimulated synovial cells, and because of its protease lability, performance on gel permeation columns, and nondialyzability, the active factor was thought to be a lowmolecular-weight protein.*, Q We termed the constellation of accelerated hyaluronate synthesis, increased glucose uptake, and increased lactate formation connective tissue activation, and the cellular mediator( s) that initiate( s ) this process was named connective tissue activating peptide (CTAP) .9 Major sources and actions of CTAP are summarized in FIGURE 1.

Modulation of the intrinsic viscosity of hyaluronic acid formed by human “fibroblasts” in vitro: The effects of hydrocortisone and colchicine

Data are presented to show that the intrinsic viscosity of hyaluronic acid elaborated into the culture medium supportinf human connective-tissue cells in vitro is modulated in a systematic manner by the cells. Intrinsic viscosity is reduced during rapid cell division and by hydrocortisone treatment. Conversely, the initial lag phase of culture growth and colchicine treatment are attended by formation of hyaluronic acid with a high intrinsic viscosity.

Effect of Treatment with AGTH or Cortisone on Anatomy of the Brain.

Summary Administration of ACTH caused chromatolysis in the cells of the paraventricular hypothalamic nucleus. Cortisone affected this nucleus but induced more widespread chromatolysis and vacuolation of thalamic and hypothalamic nerve cells.

Connective tissue activation. XXI: Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP-III) growth factors

SummaryThe quantitative radiochemical methodology described in this report allows a major increase in information generation, increased experimental flexibility, improved statistical control, and increased diversity of information per culture. Other advantages relate to economies of technical time, supplies, cells, and test materials per individual culture.Microcultures of human synovial cells incorporate [14C]glucosamine into hyaluronic acid that accumulated primarily in the media and to a lesser extent in the cell mass. CTAP-I (from lymphoid cells), CTAP-III (from human platelets), PGE2, dibutyryl cAMP, and poly(I)·poly(C) markedly stimulated hyaluronate synthesis, whereas cortisol, cycloheximide, and tunicamycin inhibited stimulated synthesis. Time studies with cycloheximide indicated that translation, essential for the activation of synovial cells, was completed by 17 h postexposure to CTAP-I. Tunicamycin also seemed to inhibit CTAP-I induced activation primarily by interpering with translation; however, tunicamycin also caused modest post-translational inhibition of hyaluronate synthesis in activated adult human synovial cells.

Production of mucopolysaccharides by synovial cells in a simplified tissue culture medium.

Summary Synovial tissue has been shown to be capable of in vitro production of a mucopolysaccharide believed to be hyaluronic acid. Cultures from donors ranging from a fetus to an elderly male shared this capacity for periods of 6 weeks to 6 months. This process is supported by a nutrient medium that is 80% defined and excludes embryo extract. Culture behavior and cellular morphology are described.

Review of united states data on neoplasms in rheumatoid arthritis

Relatively sparse literature developed during the past 30 years that sought to characterize the relationship of rheumatoid arthritis to neoplasms. The past decade has seen added concern over possible oncogenic effects of cytotoxic agents now used to manage some patients with rheumatoid arthritis. Acquisition of unambiguous data is complicated by the fact that the cumulative incidence of cancer in the general population exceeds 30 percent, and that most studies have insufficient patient numbers, duration follow-up, and attention to age, sex, race, or known etiologic agents. Thus, it is not surprising to find reports that cancer incidence is high, low, or unchanged in rheumatoid arthritis. Although equally ambiguous data were accumulated concerning potential neoplasm-inducing effects of cytotoxic drugs, concern is justified in relation to increased frequency of bladder cancer after cyclophosphamide and acute leukemia following alkylating agents.

Identification of acid mucopolysaccharides by paper chromatography

Abstract A multiple solvent paper chromatography system is described which tentatively identifies most of the known mammalian acid mucopolysaccharides, when these are examined singly. Certain mucopolysaccharides may be identified with reasonable certainty in complex mixtures of such substances. Protein contamination of significant degree does not appear to interfere with these methods. These chromatographic systems appear to be most useful for the rapid, tentative identification of acid mucopolysaccharides where only limited samples are available.

Acid mucopolysaccharide composition of serous effusions. Study of 100 patients with neoplastic and non-neoplastic conditions.

Serous effusions from 100 patients were examined for cancer cells and analyzed for acid mucopolysaccharides (AMPS) by both quantitative and qualitative methods. Strikingly high concentrations of hyaluronic acid were present not only in effusions from patients with mesotheliomas but also in effusions from many patients with other neoplasms. In 15 of 23 fluids analyzed in detail 20 to 90% of the AMPS was identified as a type of chondroitin sulfate.

Hyaluronic acid and proteoglycan synthesis by lung fibroblasts in basal and activated states

SummaryMost glycosaminoglycans (GAGs) formed by lung fibroblasts under both basal and stimulated conditions were secreted into the culture medium. High molecular weight (>106 daltons) hyaluronic acid (HA) was the major GAG species formed. Most of the SO4-GAG synthesized by lung fibroblasts was of proteoglycan (PG) monomer size. Agents stimulating complex carbohydrate formation also exhibited some selectivity with respect to whether HA or PG was formed in incremental amounts. Hyaluronate synthesis was especially stimulated by endotoxins, SO4-GAG by β-xylosides, and PG by CTAP-III.