Studies of the Levamisole inhibitory effect on rat stromal-cell commitment to mineralization

The ability of Levamisole to decrease mineralization in skeletal tissue is usually related to its effect on alkaline phosphatase (ALP). However, Levamisole is also suspected to diminish mineralization by an additional mechanism which is unrelated to the ALP control of apatite crystal growth. To delineate the time in differentiation during which Levamisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells were propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0.2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. This suggested the existence of a Levamisole-sensitive mechanism for mineralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Levamisole decreased Day 21 mineralization when added on Days 0, 3, 5, and 7, but not when added on Day 9. The Levamisole-induced inhibition of mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to mineralize is determined during the first week of culture. The early inhibitory effect of Levamisole on mineralization was associated with increased Day 12 ALP activity.     

No effects of levamisole on cytotoxic drug-induced changes of human granulopoiesis.

The effect of Levamisole on the human granulopoiesis was studied in patients randomized to receive, in addition to adjuvant chemotherapy for primary breast cancer, either no other treatment or additional unspecific immune therapy with Levamisole. The reaction of granulopoiesis to the cytostatic drugs, as characterized by changes of peripheral blood polymorphonuclear neutrophils (PMN), functional bone marrow granulocyte reserve, serial bone marrow cytology, and granulopoietic stem cells (CFU-C) in marrow and blood, was not affected by administration of Levamisole. The data support the concept that Levamisole has no direct effect on human bone marrow granulopoiesis, but that an allergic mechanism is involved in the pathogenesis of Levamisole-induced agranulocytosis. The expectation that Levamisole exerts a beneficial effect by stimulation of the granulopoiesis, as previously suggested for BCG and Corynebacterium parvum, could not be substantiated in our studies.     

Levamisole and bovine immunity: in vitro and in vivo effects on immune responses to herpesvirus immunization

Levamisole was shown to enhance in vitro blastogenic responses of bovine lymphocytes to nonspecific mitogens (phytohemagglutinin and pokeweed mitogen) as well as to infectious bovine rhinotracheitis virus and purified protein derivative. Greatest enhancement was observed at suboptimal concentrations of viral antigen. In addition to enhancing lymphocyte reactivity levamisole also affected macrophage activity as determined by increased Fc receptor activity and [3H]glucosamine incorporation. Levamisole (5-50 micrograms/mL) enhanced type II immune (or gamma) interferon production by macrophage-lymphocyte cultures. Administration of levamisole and attenuated infectious bovine rhinotracheitis vaccine virus in vivo did not elevate cellular or humoral responses.     

Dynamics of the humoral immunity indices in patients with subacute brucellosis being treated with levamisole

The dynamics of the main characteristics of humoral immunity in patients with subacute brucellosis, receiving levamisole and placebo, has been studied. Levamisole produced an immunomodulating effect manifested by an increase in the number of B-lymphocytes and a drop in the level of circulating immune complexes. Levamisole did not essentially influence the content of serum immunoglobulins and specific antibodies.     

A specific alkaline phosphatase of amphibia integument levamisole effect on short circuit current (SCC).

Using the Ussing chamber technique we have measured the short-circuit current (SCC), and so the ion transport, in the ventral skin of samples of Rana esculenta complex. The animals were not exposed to experimental treatment, and on SCC we have observed the effect of levamisole, administered either on external or internal side. Levamisole 0.0025 mM was ineffective; higher concentrations (0.025 mM, 2.5 mM), which inhibit alPase activity in tissue extracts and sections, induced an increase in SCC measurements and the effect was proportional to the concentration. Levamisole 25 mM produced a rapid and transitory increase of SCC, followed by a very quick decrement of it. Because of the action of Levamisole, "specific inhibitor of alPase activity", on ion transport in Rana skin, we propose that the alPase enzyme is probably involved in ion cutaneous transport and thus in the adaptative osmoregulation in the integument of amphibia.     

Rapid Experimental Evolution of Pesticide Resistance in C. elegans Entails No Costs and Affects the Mating System

Pesticide resistance is a major concern in natural populations and a model trait to study adaptation. Despite the importance of this trait, the dynamics of its evolution and of its ecological consequences remain largely unstudied. To fill this gap, we performed experimental evolution with replicated populations of Caenorhabditis elegans exposed to the pesticide Levamisole during 20 generations. Exposure to Levamisole resulted in decreased survival, fecundity and male frequency, which declined from 30% to zero. This was not due to differential susceptibility of males. Rather, the drug affected mobility, resulting in fewer encounters, probably leading to reduced outcrossing rates. Adaptation, i.e., increased survival and fecundity, occurred within 10 and 20 generations, respectively. Male frequency also increased by generation 20. Adaptation costs were undetected in the ancestral environment and in presence of Ivermectin, another widely-used pesticide with an opposite physiological effect. Our results demonstrate that pesticide resistance can evolve at an extremely rapid pace. Furthermore, we unravel the effects of behaviour on life-history traits and test the environmental dependence of adaptation costs. This study establishes experimental evolution as a powerful tool to tackle pesticide resistance, and paves the way to further investigations manipulating environmental and/or genetic factors underlying adaptation to pesticides.     

Effect of levamisole on binding of immune complex to mouse peritoneal macrophages

Levamisole at the concentrations of 240 and 500 micrograms/ml increased DNA-anti-DNA immune complex (IC) binding to thioglycollate-stimulated mouse (CBA) peritoneal macrophages. Reduced IC binding by macrophages of (NZB/NZW)F1 a mouse model for systemic lupus erythematosus occurs as a consequence of disease and was partially corrected after inclusion of levamisole into the reaction mixture in vitro. However, in vivo administration of 2.5 mg/kg of levamisole, the therapeutic dose, did not alter IC binding to CBA macrophages.     

Levamisole in the treatment of ascariasis in children

Levamisole (the laevorotatory isomer of tetramisole) is a new synthetic anthelmintic. A cure rate of 91% was obtained in a series of 111 ascariasis-infected children treated with a single oral dose of the drug. The failures, who were treated with the drug a second time one week later, were all cured. In a comparative study of levamisole and piperazine citrate in a series of 100 children with ascariasis a cure rate of 92% and 90% was obtained with a single dose of levamisole and piperazine respectively, indicating equal efficacy. Levamisole is better tolerated than piperazine citrate and is virtually free of toxic effects.     

Administration of levamisole to children with kidney disease exacerbation caused by recurrent respiratory infections

Levamisole (2.1-3.1 mg/kg twice a week) was administered to 6 children aged between 20 months and 6.5 years for 1 to 4 months. All children suffered form the renal diseases exacerbation (nephrotic syndrome and pyelonephritis--2 children, lipoid nephrosis and pyelonephritis--2 children, pyelonephritis with glomerular reactions--1 child, recurrent pyelonephritis--1 child) due to recurrent respiratory infections levamisole improved both clinical and biochemical parameters of renal disease. Subsequent respiratory infections were milder and less frequent. Transient decrease in thrombocyte count was seen in 4 children after 2-8 weeks of therapy with levamisole. An increase in AspAT and (or) AlAT was noted in 2 children after 1-2 months of therapy. Levamisole was withdrawn in 2 children after 30 days due to an increase in AspAT activity, prolongation of blood clotting time and thrombocytopenia.     

Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.     

Resistance to levamisole resolved at the single-channel level.

Levamisole is commonly used to treat nematode parasite infections but therapy is limited by resistance. The purpose of this study was to determine the mechanism of resistance to this selective nicotinic drug. Levamisole receptor channel currents in muscle patches from levamisole-sensitive and levamisole-resistant isolates of the parasitic nematode Oesophagostomum dentatum were compared. The number of channels present in patches of sensitive and resistant isolates was similar at 10 microM levamisole, but at 30 microM and 100 microM the resistant isolate contained fewer active patches, suggesting desensitization. Mean Po and open times were reduced in resistant isolates. The distribution of conductances of channels in the sensitive isolate revealed a heterogeneous receptor population and the presence of G25, G35, G40, and G45 subtypes. A G35 subtype was missing in the resistant isolate. Resistance to levamisole was produced by changes in the averaged properties of the levamisole receptor population, with some receptors from sensitive and resistant isolates having indistinguishable characteristics.     

Interaction of levamisole and mercaptans during thymocyte proliferation induced by concanavalin A.

Levamisole enhances[³H]thymidine uptake of murine thymocytes stimulated by concanavalin A (Con A). The proliferative response of thymocytes to Con A can also be enhanced by addition of mercaptans. Six different mercaptans were examined for this effect; three of them, 2-mercaptoethanol, cysteamine, and l-cysteine, stimulated the Con A response. Addition of levamisole to an optimal stimulatory dose of 2-mercaptoethanol or cysteamine resulted in complete inhibition of cell proliferation. Three other mercaptans, penicillamine, d-cysteine, and glutathione, failed to enhance the Con A response and, in fact, were mildly inhibitory. Levamisole gave only slightly less than normal stimulation in the presence of these mercaptans. In the absence of Con A neither levamisole nor the mercaptans stimulated cell proliferation. Oxidized 2-mercaptoethanol reacted analogously to reduced 2-mercaptoethanol both in the presence and absence of levamisole. We have interpreted these results as suggesting that the effect of levamisole is dependent upon the state of activation of the lymphocyte.     

In vitro stimulation of neutrophil motility by levamisole: maintenance of cgmp levels in chemotactically stimulated levamisole-treated neutrophils.

Levamisole at concentrations of 10(-3) M or 10(-4) M consistently increased neutrophil random motility and chemokinesis (stimulated random migration). Similar concentrations also increased directional movement of polymorphonuclear leukocytes to both endotoxin-activated serum and hydrolyzed casein. This effect on chemotaxis was due to a true stimulation and was not due solely to increased random movement. The effect of levamisole on the neutrophils could be removed by washing, but persisted if the cells were initially treated with levamisole and serum or endotoxin-activated serum. After neutrophil stimulation with chemotactic factor an initial rise in intracellular cyclic AMP levels was detected which was not influenced by prior levamisole treatment. Intracellular cyclic GMP levels after an initial slight depression, returned to resting levels and gradually diminished over a 60-minute period. Levamisole-treated cells consistently showed higher cyclic GMP levels and it is postulated that by maintaining intracellular cyclic GMP levels, microtubular assembly and cell motility might be enhanced.     

Inhibition by levamisole of metastases by cells transformed by herpes simplex virus type I.

Levamisole was tested to determine whether the drug could reduce metastases by HSV-1-transformed cells in a model hamster system. The results presented reveal an inhibition of metastases to the lungs even when the drug is inoculated after development of subcutaneous tumors at the site of inoculation of the cells.     

Levamisole augmentation of lymphocyte hyporesponsiveness to phytohaemagglutinin in patients with pulmonary tuberculosis.

Levamisole has been shown to augment the in vitro responsiveness of lymphocytes to PHA stimulation. The response of lymphocytes of 14 (42%) of 33 patients with pulmonary tuberculosis was augmented by levamisole. Augmentation was observed in 9 (64%) of 14 PHA hyporesponsives lymphocytes and in 5 (26%) of 19 normo-responsive lymphocytes.     

Effect of levamisole on alpha 2-macroglobulin and lysozyme levels in the trachea, peritoneal exudate and serum of mice

Levamisole was studied for its effect on alpha 2-macroglobulin and lysozyme content in the blood serum, peritoneal exudate, tracheal extracts of mice. Dynamics of changes in the content of these proteins was studied with its significant increase depending on the methods of the preparation administration (inhalation; intraperitoneal and per os).     

The effect of levamisole on lymphocyte protein synthesis in vitro.

Human peripheral blood lymphocytes from normal donors were studied to determine the effect of levamisole, an immunostimulating agent, on lymphocyte protein synthesis in vitro. At a concentration of 7.5 × 10^?6M, levamisole stimulation resulted in a maximal increase in protein synthesis of 136% of control. Levamisole caused no increase in protein synthesis of “T-cell depleted” populations but increased protein synthesis of “T-cell enriched” populations to a degree greater than that observed with whole lymphocyte populations. These results demonstrate that levamisole increases lymphocyte protein synthesis and suggest that the increase is due to selective stimulation of T-cells. These results provide an explanation for the effects of levamisole on lymphokine production and E-rosette formation previously reported.     

In vitro stimulation of spleen cell cultures by poly I: poly C and levamisole

Poly I: poly C and levamisole (LMS) were shown to stimulate DNA synthesis by spleen cell suspension cultures. Poly I: poly C was more effective than LMS at concentrations above 1 μg/ml; both agents were weakly stimulatory at concentrations 0.1–1.0 μg/ml. Levamisole augmented the DNA synthetic response to a supraoptimal concentration of phytohemagglutinin.     

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.     

Effect of combinations of immunomodulators on phagocytosis

The effect of levamisole, prodigiozan and lithium carbonate combinations on antimicrobial activity of blood neutrophils and peritoneal macrophages was studied on mice. The combinations were used in single doses. An increase in stimulation of phagocytosis with respect to staphylococci was observed after the use of prodigiozan in combination with lithium carbonate. Levamisole and lithium carbonate were antagonistic with respect to macrophagal phagocytosis. No advantage of the combined use of prodigiozan and levamisole to their use alone was shown.