Channing L. Hinman

Chronic Theiler's virus infection in mice: appearance of myelin basic protein in the cerebrospinal fluid and serum antibody directed against MBP

Abstract Myelin basic protein (MBP) appears frequently in the cerebrospinal fluid (CSF) of mice with chronic demyelination following intracerebral infection with Theilers murine encephalomyelitis virus (TMEV); antibody to MBP can frequently be found in the sera. The peaks of the immune responses to both MBP and TMEV coincide with the time course of the clinical signs of disease. Adsorption of mouse sera with TMEV or MBP indicate the non-identity of the antigens and the specificity of the antisera as measured by ELISA. Immunoblot analysis of sera confirmed the ELISA findings. The mechanism of induction of antibody directed against MBP and its role in TMEV-associated demyelination remain to be determined.

Activities of cobra venom cytotoxins toward heart and leukemic T-cells depend on localized amino acid differences

Several studies have suggested that along the concave surface of cobra venom cytotoxins, a hydrophobic region flanked by positively charged amino acid side-chains, as well as by tyrosine and/or serine/threonine, allows these toxins to depolarize muscle or cause cytolysis. Comparison of biological activities among structurally homologous toxins, however, has revealed significant functional diversity. The objective of the present study was to examine several toxins purified from different cobra venoms with regard to their ability to bind to and kill human T-lymphocytes and rat heart cell myoblasts. The activities observed were then correlated with differences in amino acid residues which occur in restricted regions of the toxins. The absence of an aromatic residue at position 11 (Loop 1) resulted in a lower cytolytic response at every concentration tested. A simple inversion of two residues in the amino acid sequence of toxin Loop 3 selectively impaired heart cell binding and cytolysis, but had no effect on T-cells. Loss of a positively charged residue in the tip of Loop 2 minimally affected binding but significantly reduced cytolysis. Replacement of valine at positions 27 and 32, along with the introduction of a negative charge at the tip of Loop 2, interfered with binding to either cell type and caused a reduction in cytolysis. The results of this study suggest that no one loop or region is solely responsible for the toxins biological activity. However, because the binding and cytolytic sites within these toxins are distinct, it may become possible to develop toxin derivatives in which only selected activities are enhanced.

Effects of cardiotoxin d from naja naja siamensis snake venom upon murine splenic lymphocytes

Cardiotoxin D from Naja naja siamensis is cytotoxic to T-lymphocytes above 150 femtomoles/10(6) cells. Equivalent lysis of macrophages or B-lymphocytes requires at least 1000 times more toxin. Reduction and carboxamidomethylation of cardiotoxin D does not effect T cell lysis. At higher toxin concentrations, 50% T-cell lysis occurs within 10 min. Splenocytes cultured with mitogens are up to five times more susceptible to toxin than unstimulated cells. Cardiotoxin D may directly disrupt the plasma membrane, since lysis is unaltered at 4 degrees C.

Selective cytolysis by a protein toxin as a consequence of direct interaction with the lymphocyte plasma membrane

Two lines of evidence support the concept that cardiotoxin from Naja naja siamensis interacts directly with the plasma membrane to produce selective cytolysis of lymphocytes. Toxin adsorbed to the wells of microtiter plates retained the ability to lyse murine T-lymphocytes, but lost the ability to destroy natural killer cells, whereas soluble toxin obliterated both cell types. Second, toxin covalently coupled to 100-microns-diameter agarose beads, such that endocytosis would be precluded, effectively lysed L1210 tumor T-lymphocytes. Although differences were observed among susceptibilities of a variety of mouse and human tumor lymphocyte cell lines to toxin-mediated lysis, these differences were not so great as the differences between tumor and normal lymphocytes. The intrinsic selectivity of the toxin for T-lymphocytes, plus its retention of cytolytic potential when affixed to a solid support, suggests that such a protein could be applied therapeutically. In addition, based upon activity which is temperature-independent and not influenced by the absence or presence of external calcium, it appears that the toxins mode of action may be different from that involved with erythrocyte hemolysis or with skeletal or cardiac muscle depolarization.

A membrane-lytic immunoconjugate selective for human tumor T-lymphocytes

An immunoconjugate was constructed from a monoclonal antibody recognizing human T-lymphoblastoid cells and a membrane-lytic cytotoxin purified from the venom of the Thailand cobra. Activities of this novel immunoconjugate were compared using human and murine T-lymphocyte cell lines. The ability of the conjugate to inhibit human T-cell proliferation, as measured by incorporation of [3H]thymidine, was three to four times higher than its ability to inhibit proliferation of mouse L1210 cells. The immunoconjugate EC50 for human CEM cells was equivalent to 0.1 nmoles per 2 x 10(5) target cells. Immunoconjugate selectivity paralleled the monoclonal antibodys binding characteristics. Preincubation with free antibody blocked the effect of the conjugate, but only upon the human target cells. This study supports the feasibility of directing a toxic moiety to the surface of a cancer cell to accomplish cell destruction without requiring prior toxin internalization and uncoupling from its antibody carrier.

Apoptotic activities in closely related styryllactone stereoisomers toward human tumor cell lines: Investigation of synergism of styryllactone-induced apoptosis with TRAIL.

A related series of styryllactones with small functional and stereochemical variations were compiled for a comparative study of their apoptotic activities toward two tumorigenic and one non-tumorigenic control cell line. While a substantial range of intrinsic activity was observed, the relative order of activity of the different compounds toward the cell types varied somewhat as did the relative ratios of apoptosis and necrosis observed in conjunction with the loss of cell viability. While some of the styryllactones showed substantial activity, a small but significant apoptosis-induced synergism was demonstrated with (-)-altholactone and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).

Experimental allergic encephalomyelitis in mice: presence of myelin basic protein in cerebrospinal fluid

The concentration of myelin basic protein (MBP) in cerebrospinal fluid (CSF) correlates with the development of experimental allergic encephalomyelitis following intradermal injection with encephalitogen in adjuvant; MBP is absent in controls inoculated with adjuvants only. The presence of MBP is a sensitive indicator of disease inasmuch as CNS-inoculated mice with neurologic signs had an average of 0.29 ng/microliter of MBP in their CSF and controls, including normal or adjuvants only, had an average of 0.03 ng/microliter. The amount present per microliter of CSF, as well as the absolute amount, obtained from an individual mouse do not always reflect the severity of disease as indicated by clinical signs and the pathology observed in a sampling of the neuraxis. The presence of MBP does correlate with demyelination, although the extent of pathology observed by light microscopy in the mouse model is minimal, associated only with the inflammatory response, and does not extend beyond the zone of the perivascular cuff.

Ablation of natural killer cell function by soluble cardiotoxin

A one-hour preincubation of nonadherent murine spleen cells with a soluble membrane-active cardiotoxin purified from the venom of the Thailand cobra Naja naja siamensis results in the destruction of natural killer (NK) cell activity against YAC-1 target cells in a dose-dependent manner. Prior in vivo induction of interferon production by polyinosinic/polycytidylic acid does not avert the cardiotoxin inhibition of NK function. Loss of complement-mediated lysis of cells capable of binding an NK-1.1 monoclonal antibody suggests that the cardiotoxin directly affects the integrity of the NK cell plasma membrane. Cardiotoxin which has been adsorbed to the surface of polystyrene tissue culture plates retains the ability to lyse splenic T lymphocytes, but loses the ability to interfere with NK activity, as measured either by the release of 51Cr or by the uptake of 3H-thymidine by the target lymphoma cells, suggesting that different parts of the cardiotoxin molecule are responsible for destruction of the two types of lymphocytes.

In-Line Affinity Chromatographic Removal of Specific Antibody from Rabbits with Experimental Myasthenia Gravis as A Prelude to Immunotherapy

Myasthenia gravis is an autoimmune disease in which antibodies are produced against ones acetylcholine receptors, resulting in complement-mediated membrane destruction and internalization of antibody-receptor complexes. Symptoms range from weakening of extraocular muscles to severe impairment of movement and breathing. Prior to administering a therapeutic agent to eliminate antibody-producing lymphocytes, it will be necessary to remove specific antibody from the circulation. This process was investigated in an animal model of ex vivo specific immunoadsorption using awake, conscious rabbits. Following arterial blood separation, plasma was pumped upward through an affinity column containing covalently-bound acetylcholine receptor. Treated plasma was returned to the rabbit. Within a one-hour ex vivo procedure, specific antibody levels could be lowered from 16.2 ng/ml to less than 0.6 ng/ml, a reduction of more than 95%. By washing the column, at least four exchanges could be performed before specific antibody removal significantly diminished. The effects of specific antibody removal upon muscle function varied among individual rabbits, but if symptoms were not severe following passive transfer of purified monoclonal antibody to induce myasthenia, removal of 60% of the total specific antibody resulted in clinical improvement, as monitored by an animals response to gallamine triethiodide.

Prostatic involution after intraprostatic injection of cobra toxin.

PURPOSE We evaluated the comparative effects of intraprostatic injection of cobra cardiotoxin D and botulinum toxin type A on prostate structure in the rat model. MATERIALS AND METHODS A total of 18 Sprague-Dawley® rats weighing 500 to 600 gm received a single 0.1 ml injection of saline (6), botulinum toxin type A (6) or the cardiotoxin D (6) component of cobra (Naja naja atra) toxin in the right and left ventral lobes of the prostate. At 14 days the rats were sacrificed. The prostate glands were harvested, weighed and processed for immunohistochemical and morphological studies. RESULTS Prostate glands injected with cardiotoxin D showed significantly decreased weight compared to that of prostates injected with botulinum toxin type A and the saline control. Prostatic atrophy in the glandular component with flattening of the epithelial lining was seen histologically in rats that received botulinum toxin and cardiotoxin D. Each group injected with cardiotoxin D and botulinum toxin showed a significant increase in the number of apoptotic cells compared with controls while only the botulinum toxin group showed a significant increase in the number of proliferating cells. Only rats injected with botulinum toxin had body weight loss. CONCLUSIONS Our study shows that intraprostatic injection of cobra cardiotoxin D induces prostatic atrophy and leads to a decrease in prostatic weight greater than that of intraprostatic injection of botulinum toxin type A. No systemic effects, such as decreased body weight, were noted after cardiotoxin D injection. Further studies are warranted but the statistically significant decrease in the number of proliferating cells implies a prolonged effect of cardiotoxin D.