Irene J. Check

A comparison of commercially available adjuvants for use in research

We evaluated an adjuvant, TiterMax, as an alternative to complete Freunds adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

Inflammatory meningeal masses of unexplained origin. An ultrastructural and immunological study.

Two patients with inflammatory meningeal masses were studied. Lesions in both patients showed varying proportions of meningothelial and inflammatory components. The non-neoplastic nature of the inflammation was confirmed in one case by lymphocyte surface marker study, which showed T and B cells in one to four ratio, and by immunohistochemistry, which revealed polyclonal plasma cells. The abundant histiocytes contained muramidase and often enclosed intact lymphocytes or plasma cells within their cytoplasm, i.e., emperipolesis. Their surfaces bore slender interdigitating pseudopodia, intercellular junctions, and subplasmalemmal linear densities. The derivation of these histiocytes is uncertain: mononuclear phagocytes, meningothelial cells, and multipotential meningeal cells are all possible progenitors. A comparison with eleven similar reported cases reveals a tendency for inflammatory meningeal masses to occur in the young, as well as a predilection for posterior fossa involvement. They resemble the extranodal lesions of sinus histiocytosis with massive lymphadenopathy, as well as plasma cell granulomas or inflammatory pseudotumors of lung and other tissues. However, it is possible that these lesions represent a variant of meningioma in which an unusual immunological response has been evoked.

Modification of acute focal ischemia in rabbits by poloxamer 188

We studied the effect of a synthetic copolymer surfactant, poloxamer 188, on cerebral blood flow in a rabbit model of focal cerebral ischemia. Following retro-orbital craniectomy, the parietal branch of the middle cerebral artery was occluded with bipolar current. Cerebral blood flow was measured by the hydrogen clearance technique using platinum-iridium electrodes placed within the parietal cortex. Ten rabbits were infused with 50 mg/kg poloxamer 188 in saline beginning 30 minutes after occlusion; 12 control rabbits received an equal volume of saline. Poloxamer 188 increased blood flow significantly in areas of severe or moderate ischemia but had little effect in areas with mild or no ischemia. The improvement in blood flow could not be accounted for by hemodilution, and the copolymer did not affect blood viscosity at any shear rate from 1 to 100 sec-1. We hypothesize that poloxamer 188 increases circulation in ischemic tissue by inhibiting adhesive interactions among proteins (fibrin and fibrinogen) and cells in the microcirculation.

The Effect of Poloxamer 188 on the Rate of In Vitro Thrombolysis Mediated by t-PA and Streptokinase

Poloxamer 188 is a nonionic block copolymer surfactant which is undergoing clinical trials as a haemorheologic agent. A simulated thrombus preparation was designed to evaluate its effects on fibrinolysis and thrombolysis under flow conditions similar to those which might be encountered within an occlusive coronary thrombus. 125I labelled human fibrinogen (20 μCi/ml) was mixed with platelet-poor citrated plasma, recalcified, and allowed to clot for 1 h at 37°C in a tuberculin syringe filled with small glass beads. This produced a fibrin clot permeated with microscopic channels simulating those of a recently formed thrombus. After washing the plasma bead clots for 30 min with normal saline, heparinised blood containing tissue plasminogen activator (t-PA) or streptokinase, with and without 0.1% poloxamer 188, was added to the columns and rates of flow and elution of 12 125-fibrin degradation products were measured. Poloxamer 188 alone had no ability to lyse fibrin. However, it increased the rate at which t-PA or streptokinase caused lysis of simulated thrombi by as much as 4-fold. This accelerated lysis was associated with up to a 20-fold increase in flow through the thrombus and with elution of larger fragments of fibrin, but not with increased plasmin activity as assessed with a colourimetric substrate. Consequently, poloxamer 188 appeared to accelerate thrombolysis without directly affecting any of the components of the fibrinolytic system. We propose that the ability of poloxamer 188 to modulate thrombolysis is due to its haemorheologic properties.

Nonionic Block Copolymer Surfactants as Immunological Adjuvants: Mechanisms of Action and Novel Formulations

Modern molecular biology has provided us with the ability to produce custom tailored antigens with an ease undreamed of a few years ago. However, experience has also shown that most nonreplicating purified subunit antigens are weak immunogens that will require immunopotentiation to become the hoped for effective vaccines of the future. The dominant paradigm of modern immunology is that immunogenicity and immune responses are controlled by specific interactions between various ligands and their receptors. From our perspective, producing effective adjuvants for subunit vaccines will require looking beyond this paradigm to a consideration of nonspecific physicochemical factors which subtly but powerfully influence immune responses.

Serum protein changes during cardiopulmonary bypass: implications for host defence:

A reduction in host defence capability, with increased risks of infection, occurs after open-heart surgery to a greater degree than after nonbypass surgery. Our costs for postoperative infections are large, about 15% of the total cost of open-heart surgery; better understanding of factors contributing to infection might allow reduction in these costs. Disturbance of the mechanisms involved in the handling of haemoglobin and iron may be among such factors, since free iron is a facultative requirement for the growth of all micro-organisms. We evaluated the change in serum proteins, particularly haptoglobin, during cardiopulmonary bypass. This study, in eight patients undergoing uncomplicated aortocoronary bypass employing a bubble oxygenator with haemodilution, and without clinical evidence of haemolysis, showed that free, unbound haptoglobin, evaluated by agarose gel electrophoresis, disappeared and was replaced by haemoglobin-bound haptoglobin within minutes of going on bypass. Such changes were not seen in three control patients who underwent comparably extensive surgery without bypass or in-bypass controls, in whom blood was evaluated after dilution with oxygenator prime. The disappearance of free haptoglobin and the possible disturbance of iron transport may have implications for host defences after bypass.

Hypocomplementemic Proliferative Glomerulonephritis with C3 Nephritic-Factor-Like Activity in Multiple Myeloma

Advanced renal failure, nephrotic-range proteinuria due to proliferative glomerulonephritis and multiple myeloma with circulating IgG2 lambda and free lambda light-chain paraproteins occurred in a 31-year-old male. Commonly established causes of renal failure in multiple myeloma were excluded. Immunofluorescence revealed heavy granular glomerular deposition of C3. Serum C3 was decreased, and C3c was increased. C3 nephritic-factor (C3 NeF)-like activity was demonstrated in the serum. Plasmapheresis and chemotherapy resulted in a decrease in paraprotein concentration up to 90%, a decrease in C3 NeF-like activity to negligible, normal serum complement levels and a marked improvement in both renal function and proteinuria. With reference to the literature, the possibility of a syndrome of paraproteinemia, C3 NeF-like activity and glomerulonephritis is forwarded.

Immunoendocrine Modulation and Stimulation of Hematopoiesis with the Ionophore Copolymer T150R1

Abstract T150R1, an 8000-dalton copolymer with sodium ionophore activity, has been shown to modulate cellular responses in multiple systems. In this article, we studied its effects on lymphoid and hematopoietic organs in the context of the adrenal-pituitary axis. When injected in mice as an oil in water emulsion, T150R1 caused a rapid, profound, and dose-dependent thymic involution accompanied by splenic hyperplasia. Time course experiments with a 2.5-mg dose revealed that the thymus size was minimal at Day 2, rose to normal by Day 14, then enlarged and gradually returned to normal by Week 6 postinjection. Thymic involution was due to cellular depletion of the cortical area, whereas thymic enlargment was due to cortical hyperplasia. Splenomegaly was seen as early as Day 4, peaked by Day 14, and gradually returned to normal by Week 6. The splenic enlargement was due to hyperplasia of the red pulp, with evidence of proliferating erythropoietic, myelopoietic, and megakaryopoietic precursors. In addition, the bone marrow was stimulated and extramedullary hematopoiesis was present in the liver. The effects of T150R1 on the thymus appeared to be mediated by corticosteroids while the effects on hematopoiesis were not. Corticosterone and ACTH levels were increased in treated animals. Adrenalectomy diminished the T150R1-induced thymic involution but enhanced the splenic hyperplasia. Hypophysectomy did not prevent thymic involution, suggesting that T150R1 has endocrine stimulatory effects. These data suggest that T150R1 represents a new class of ionophores which may act on excitable cells within the endocrine, immune, and hematopoietic systems.

Systemic and lung protein changes in sarcoidosis. Lymphocyte counts, gallium uptake values, and serum angiotensin-converting enzyme levels may reflect different aspects of disease activity.

BAL lymphocyte percentages, quantitated gallium-67 lung uptake, and SACE levels have all been proposed as measures of disease activity in sarcoidosis. We analyzed 32 paired sera and BAL fluids from sarcoidosis patients by high-resolution agarose electrophoresis to look for protein changes characteristic of systemic or local inflammation and compared the results with those from the above tests. Nine patients (group 1) had serum inflammatory protein changes and increased total protein, albumin, beta 1-globulin (transferrin), and gamma-globulin levels in fluid recovered by BAL. Thirteen patients (group 2) had normal protein levels in sera but abnormal protein levels in BAL specimens. Ten patients (group 3) had normal protein levels in sera and in BAL specimens. Patients in groups 1 and 2 had a disproportionate increase in beta 1-globulin (transferrin) and gamma-globulin levels in their BAL specimens. The BAL lymphocyte percentage changes paralleled the BAL protein level changes, suggesting relationships among the immunoregulatory role of these cells, increased local immunoglobulin synthesis, and the pathogenesis of altered alveolar permeability. Gallium-67 uptake was highest in patients with serum inflammatory protein changes. Thus, systemic inflammation may facilitate pulmonary gallium-67 uptake, possibly by changes in BAL fluid or serum transferrin saturation and/or kinetics. SACE levels showed no relationship to changes in the levels of serum or BAL proteins. These data suggest that the various proposed measures of disease activity reflect different aspects of inflammation in sarcoidosis.

Immunomodulatory lonophore Copolymers, T150R1 and T130R2, Induce Corticotropin from Anterior Pituitary Cells

Abstract T150R1 is a synthetic copolymer with Na+ ionophore activity. We demonstrated previously that T150R1, when injected into mice, produces rapid thymic involution with depletion of cortical thymocytes. Elevated serum ACTH and corticosterone levels, as well as abrogation of the effects of T150R1 on the thymus by adrenalectomy and hypophysectomy, suggested a pituitary-mediated mechanism. In this work, we investigated the ability of T150R1, and of the related ionophore copolymer T130R2, to stimulate ACTH in vitro from the mouse anterior pituitary cell line AtT-20. Copolymer-induced ACTH release was dose-, time-, and temperature-dependent. Hormone induction peaked at 30°C for T150R1 and 37°C for T130R2. The temperature dependence of ACTH release paralleled that of ionophore activity measured in red blood cells, providing evidence that the ability to induce ACTH is related to the ionophore property of the copolymers. Peak ionophore activity and hormonal release occurred at the temperatures when the copolymers form partially soluble complexes which interact optimally with cell membranes. Cotreatment with exogenous phospholipase C inhibited the effects of T150R1, which suggests that the enzyme either blocks the insertion of T150R1 into the cell membrane or that the phospholipase C-induced increase in intracellular calcium inhibits the ionophore activity of T150R1. These data support an ionophore mechanism for copolymer-induced ACTH release in which changes in the physicochemical structure of the copolymers may affect their interaction with cell membranes. The data also suggest that direct stimulation of pituitary ACTH accounts for at least some of the in vivo immunomodulatory effects of T150R1.