Neutrophil extracellular traps as a potential source of autoantigen in cocaine-associated autoimmunity.

Objective: Exposure to illicit cocaine and its frequent adulterant, levamisole, is associated with ANCAs targeting neutrophil elastase (NE), neutropenia and vasculitic/thrombotic skin purpura. The mechanisms of cocaine/levamisole-associated autoimmunity (CLAA) are unknown. The aim of this study was to assess the ability of cocaine and levamisole to induce the release of neutrophil extracellular traps (NETs), a potential source of autoantigen and tissue injury. Methods: We performed quantitative and qualitative assessment of NET formation in neutrophils from healthy donors exposed to either drug in vitro . In addition, IgG from sera of individuals with CLAA (CLAA-IgG) was assessed for its ability to enhance formation of, and to bind to, drug-induced NETs. Results: Both cocaine and levamisole could induce formation of NETs enriched in NE and, potentially, inflammatory mitochondrial DNA. Both drugs could also augment simultaneous release of B cell-activating factor belonging to the TNF family (BAFF). CLAA-IgG, but not IgG from healthy individuals, could potentiate drug-induced NETosis. Furthermore, CLAA-IgG, but not ANCA + control IgG, bound to drug-induced NETs in a pattern consistent with NE targeting. Conclusion: Both cocaine and levamisole may contribute to the development of ANCAs by inducing release of potentially inflammatory NETs in association with NE autoantigen and BAFF. Enhancement of drug-induced NET release by CLAA-IgG provides a potential mechanism linking vasculitis/pupuric skin disease to acute drug exposure in patients with CLAA. Further study of this under-recognized form of autoimmunity will be likely to provide mechanistic insight into ANCA-associated vasculitis and other diseases associated with NETosis.

Levamisole enhances immunity in ducklings vaccinated against Riemerella anatipestifer.

Oil-adjuvant-inactivated vaccine is one of the most cost-effective vaccines used to protect ducklings against RA infection; however, it does not provide complete protection in very young ducklings with immature immune systems. In the current study, LMS was used as an immunopotentiator to improve the immune system in ducklings. Serum immunoglobulin (Ig)G titers and the secretions of both Th1-type (IFN-γ and IL-2) and Th2-type (IL-4 and IL-10) cytokines were higher in ducklings that had been vaccinated with LMS. In addition, a significantly higher T-lymphocyte proliferation rate was obtained with the addition of LMS. Furthermore, all of the ducklings vaccinated with LMS were protected against RA on the 9th day post-vaccination, whereas only 69.2% of the ducklings were protected in the group that did not receive LMS. These results suggest that LMS might be a useful adjuvant to enhance the immune response of ducklings. The use of LMS may also alleviate local injection lesions, caused by the oil-emulsion vaccine, by reducing the dose of the vaccine.

Cocaine-induced vasculitis: clinical and immunological spectrum.

Levamisole-contaminated cocaine has recently been recognized in North America and Europe, and its use is associated with a variety of clinical and autoimmune abnormalities. The clinical characteristic seems to be the presence of a painful purpuric skin rash that predominantly affects the ear lobes and cheeks, often accompanied by systemic manifestations including fever, malaise, arthralgias, myalgias, and laboratory abnormalities, for example leukopenia, neutropenia, positive ANA, ANCA, and phospholipid antibodies. Most of these manifestations can be seen with the use of either drug, especially levamisole. There is no specific therapy, and discontinuation of its use is followed by improvement. Prednisone and immunosuppressive therapy may be needed at times. Further use of the drug is characterized by recurrence of most of the complaints.

Meta-analysis: levamisole improves the immune response to hepatitis B vaccine in dialysis patients.

BACKGROUND: Patients undergoing maintenance dialysis often fail to mount protective antibodies to hepatitis B virus surface antigen (HBsAg) following vaccination against hepatitis B virus (HBV). Some authors have suggested that levamisole improves immune response to HBV vaccine in dialysis population. However, consistent information on this issue does not exist. AIM: To evaluate efficacy and safety of levamisole as adjuvant to hepatitis B virus (HBV) vaccine in dialysis patients by performing a systematic review of the literature with a meta-analysis of clinical trials. METHODS: We used the random-effects model of DerSimonian and Laird, with heterogeneity and sensitivity analyses. Only trials comparing the seroresponse rate in study subjects (levamisole plus HBV vaccine) vs. controls (HBV vaccine alone) were included. The end point of interest was the rate of patients showing seroprotective anti-hepatitis B titres at completion of HBV vaccine schedule in study vs. control groups. RESULTS: We identified four studies involving 328 unique patients on regular dialysis. Only prospective, randomized clinical trials (RCTs) were included. Pooling of study results showed a significant increase in response rates among study (levamisole plus HBV vaccine) vs. control (HBV vaccine alone) patients; the pooled Odds Ratio was 2.432 (95% Confidence Intervals, 1.34; 4.403), P = 0.002. No study heterogeneity was found. These results did not change in various subgroups of interest. CONCLUSIONS: Our meta-analysis showed that levamisole significantly improves immune response to hepatitis B vaccine in dialysis population. The limited number of patients precluded more conclusions.

Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac nonenterotoxigenic E. coli strain.

Colidiarrhea and colienterotoxemia caused by F4(+) and/or F18(+) enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac(+) non-ETEC vaccine candidate strain against challenge infection with F4ac(+) ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac(+) non-ETEC strain (at day 0), and challenged with F4ac(+) ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3(+), CD45RA(+), CD45RC(+), CD21(+), IgA(+) and myeloid SWC3(+) cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer's patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3(+), CD45RC(+) and SWC3(+) cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21(+) cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac(+) non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.

Enhancing effects of the chemical adjuvant levamisole on the DNA vaccine pVIR-P12A-IL18-3C.

DNA-based vaccination is an attractive alternative for overcoming the disadvantages of inactivated virus vaccines; however, DNA vaccines alone often generate only weak immune responses. In this study, the efficacy of LMS as a chemical adjuvant on a DNA vaccine (pVIR-P12A-IL18-3C) encoding the P1-2A and 3C genes of the FMDV and swine IL-18, which provides protection against FMDV challenge, was tested. All test pigs were administered booster vaccinations 28 days after the initial inoculation, and were challenged with 1000 ID50 FMDV O/NY00 20 days after the booster vaccination. Positive and negative control groups were inoculated with inactivated virus vaccine and PBS respectively. The DNA vaccine plus LMS induced greater humoral and cell-mediated responses than the DNA vaccine alone, as evidenced by higher concentrations of neutralizing and specific anti-FMDV antibodies, and by higher concentrations of T-lymphocyte proliferation and IFN-y production, respectively. FMDV challenge revealed that the DNA vaccine plus LMS provided higher protection than the DNA vaccine alone. This study demonstrates that LMS may be useful as an adjuvant for improving the protective efficiency of DNA vaccination against FMDV in pigs.

Synergistic effects of adjuvants interferon-gamma and levamisole on DNA vaccination against infection with Newcastle disease virus.

Both humoral and cell-mediated immune responses are important to protect animals from initial acute viral infection and establishment of chronic infection. Adjuvants for DNA vaccines can influence the balance between humoral and cell-mediated immunities. In this study, a DNA vaccine encoding the hemagglutinin-neuraminidase and fusion genes of Newcastle disease virus (NDV) incorporated with chicken interferon(provax-chIFN-gamma) cDNA as a molecular adjuvant and levamisole (LMS) as a chemical adjuvant was tested for its efficacy in protection against NDV lethal challenge. Compared with DNA vaccine alone, the DNA vaccine with provax-chIFN-gamma plus LMS induced significantly higher humoral and cell-mediated responses, as shown by higher levels of hemagglutination inhibition (HI) titers and T cell proliferation. In addition, the DNA vaccine with provax-chIFN-gamma plus LMS formulation increased the expression of IFN-gamma, interleukin (IL)-2, IL-4, IL-12, and IL-13, suggesting that the effectiveness of the IFN-gamma and LMS formulation is partly due to the enhancement of balanced cytokine production. Furthermore, the two adjuvants yielded 80% protection in chickens against challenge with a lethal dose of the virulent NDV strain. This study demonstrates that the synergistic effects of provax-chIFN-gamma plus LMS as the adjuvants in NDV DNA vaccination could be used to improve protective efficacy in chickens.

Induction of alkaline phosphatase in the inflamed intestine: a novel pharmacological target for inflammatory bowel disease.

This study demonstrates the upregulation of alkaline phosphatase and the mechanisms involved in experimental colitis. All models of ileal and colonic inflammation examined, which were characterized by significant oxidative stress and neutrophil infiltration, resulted in an increase in alkaline phosphatase activity which was attributable to both epithelial cells and cells of the lamina propria, mainly leukocytes. The increase in alkaline phosphatase sensitivity to the inhibitors levamisole and homoarginine, together with changes in the apparent molecular size and in the sialization of the enzyme, indicated a change in the isoform expressed. An increase in tissue non-specific alkaline phosphatase expression was observed by Western blotting. Treatment with the bone/kidney alkaline phosphatase inhibitor levamisole or a monoclonal antibody resulted in significant protection from colonic inflammation. Taken together, these results indicate that the kidney isoform is a marker of intestinal inflammation and that it might even constitute a target for pharmacological intervention.

Total serum immunoglobulin M levels are affected by immunomodulators in seabream (Sparus aurata L.).

Immunoglobulin M (IgM) is a major component of the teleost humoral immune system. Despite the significance of IgM levels as an immune parameter, there are relatively few studies on changes induced in its total levels in serum. This study examines the effects of several immunomodulators (vitamin A, chitin, yeast cells or levamisole, which act as immunostimulants, and crowding, hypoxia or anaesthetics, which act as stressors) upon the total serum IgM levels of non-immunized gilthead seabream (Sparus aurata L.). Total serum IgM levels of fish fed with the assayed immunostimulant-supplemented diets were statistically higher than those in fish fed a non-supplemented diet, especially in the case of levamisole. On the other hand, serum IgM levels of fish subjected to different stressors were not affected by crowding, hypoxia or certain anaesthetics. However, benzocaine and a narcotic dose of 2-phenoxyethanol provoked a great reduction, while quinaldine sulphate increased IgM levels to a significant degree. These results show how the seric IgM levels can be differently affected by some immunomodulators and the important role they may play in the regulation of total circulating IgM levels in seabream. The possibility of using total serum IgM for assessing immunostimulation, disease diagnosis and stress symptoms during fish farming is discussed.

Levamisole is a potent enhancer of gilthead seabream natural cytotoxic activity.

Gilthead seabream (Sparus aurata L.) head-kidney (HK) leucocytes were incubated with 10(3) to 10(-4) ng levamisole/ml for 4, 24 or 48 h and then assayed for their natural cytotoxic activity against xenogeneic tumor cells. This activity was slightly increased after 24 h of incubation. In a second experiment, fish specimens were fed 0, 75, 150 or 300 mg levamisole/kg diet for 10 consecutive days. The fish were then fed a commercial non-supplemented diet and sampled 0, 1, 2, 3, 4 and 6 weeks post-administration of levamisole. The cytotoxic activity was found increased along the experiment and remained greatly enhanced at the end. In conclusion, levamisole enhanced seabream natural cytotoxic cell activity both in vitro and in vivo and had a great and lasting action when administered by feeding.

Is oral levamisole immunostimulation in rats mediated by reduced levels of free plasma corticosterone?

A brief review of the immunostimulative and anti-inflammatory properties of levamisole is followed by a research report which indicates that in rats under antigenic challenge by ovalbumin, oral levamisole (18 mg/kg) decreases corticosterone levels. This previously unreported finding may belie the immunostimulative effect of levamisole.

Immunologic effects of levamisole in mice and humans: evidence for augmented antibody response without modulation of cellular cytotoxicity.

Selected immunomodulatory effects of levamisole were studied in patients with asymptomatic metastatic colon cancer and in a preclinical model (CF1 female mice treated with methyl-azoxymethanol acetate) for colon tumors. In the patient population studied, there was no augmentation of cellular cytotoxicity or alteration in lymphocyte subpopulations that participate in these functions. An increase in Fc receptor binding on circulating monocytes was apparent at the 4-week timepoint; however, a corresponding increase in antibody-dependent cellular cytotoxicity was observed in only one of the six patients studied. In most patients, cellular cytotoxicity diminished with time. No significant effects on cellular immunity or carcinogenesis were observed in our murine studies. However, treatment with levamisole did increase circulating immunoglobulin levels and IgM response in mice immunized with the T-dependent antigen keyhole limpet hemocyanin. This parameter was not tested in the human trial. Failure to demonstrate antitumor effects on cellular immunity by levamisole in both human and murine studies suggests that these effects, if they do exist, may involve immunological parameters that were not tested using our methods or that may not be apparent in patients with more advanced malignancy.

Levamisole and its influence on the immune response of lambs.

Ten parasite-free lambs were drenched with 8 mg/kg of levamisole on days 0 and 28 and were injected with human erythrocytes and ovalbumin one day after each drench. Ten other antigen-injected lambs were not drenched with anthelmintic as controls. Lymphocytes from the control and drenched lambs were cultured in vitro with RPMI 1640 plus 5% fetal calf serum (FCS), with 50% autologous serum only, with concanavalin A (Con A) or with phytohaemagglutinin (PHA). Decreased blastogenesis was observed in cells from the drenched lambs cultured in the presence or absence of mitogen and was most obvious when 50% autologous serum was used, particularly with PHA, and when lymphocytes were collected 3 and 7 days after the first and 3 days after the second antigen injection. There were no significant changes in antibody titres between the groups. Decreased serum complement activity was seen 3 days after the second antigen injection in the drenched lambs. Although there was a significant reduction in the serum insulin-like growth factor I levels 4 days after each levamisole drench, the drenched lambs gained significantly more weight than the non-drenched control lambs.

Preliminary development of a live drug-controlled vaccine against bovine babesiosis using the Mongolian gerbil, Meriones unguiculatus.

This study investigated the practicality and potential of the gerbil, Meriones unguiculatus, as a source of live Babesia divergens vaccine and also as a model for the use of the vaccine in cattle. A series of experiments with gerbils concerning vaccine infectivity, immunogenicity and safety were carried out. It was concluded that the use of RPMI medium/40% foetal calf serum as a diluent improved vaccine infectivity, but that the parasitaemia of the blood obtained from donor gerbils had little or no effect. The immunostimulants levamisole and killed Corynebacterium parvum improved vaccine immunogenicity and it was also shown that the subcutaneous route of infection resulted in the greatest host response. Control of vaccine virulence with drugs was only possible when drugs with prophylactic properties, such as imidocarb and long-acting oxytetracycline, were used. More studies are required on all these topics, particularly with regard to their applicability to cattle, and also concerning the possible attenuation of the parasite by manipulation in the gerbil host.

Levamisole, the story and the lessons.

The history of the use of levamisole in man is summarized, from its start as an anthelmintic in the early sixties, through its world-wide recognition as an immunotropic agent especially in the seventies and early eighties, and its return to clinical prominence in 1989-90 as an effective adjuvant treatment for operable colon cancer. The knowledge accumulated from experimental tumour models and from clinical use in various types of cancer, supplemented with the recent evidence obtained from large-scale controlled trials in resectable colon cancer is reviewed. It is speculated that we may not have seen the end of levamisole story yet; also, the role of serendipidity in drug research is emphasized.

Levamisole in the adjuvant treatment of colon cancer.

The chemistry, pharmacology, pharmacokinetics, assay methodologies, adverse effects, and dosage of levamisole are described, and the clinical studies of levamisole therapy in patients with colorectal carcinoma are reviewed. Levamisole is a synthetic, orally active agent that has antihelmintic and immunomodulatory properties. It is capable of inducing T-cell differentiation and restoring depressed effector functions of peripheral lymphocytes and phagocytes to normal. The drug is well absorbed from the gastrointestinal tract after oral administration and is extensively metabolized by the liver. Gas chromatography and high-performance liquid chromatography are the most common methods used to measure concentrations of levamisole in biologic fluids. Levamisole combined with fluorouracil has been associated with a one-third reduction in recurrence and risk of death in patients with surgically resected Dukes stage C colon cancer; this combination is now recommended as standard therapy in these patients. Uses in patients with rectal carcinoma, Dukes stage B colon cancer, metastatic colon cancer, other malignancies, or nonmalignant disorders remain investigational. Common adverse effects include nausea, abdominal pain, vomiting, diarrhea, metallic or altered taste, flulike symptoms, mood elevation, insomnia, hyperalertness, dizziness, and headache. The most serious adverse effect associated with levamisole is granulocytopenia. The FDA-approved dosage of levamisole is 50 mg orally every eight hours for three days every two weeks. Levamisole therapy is to be initiated no earlier than 7 and no later than 30 days after surgery and is to be continued for one year. Levamisole combined with fluorouracil has been associated with a one-third reduction in recurrence and risk of death in patients with resected stage C colon cancer. Further research is needed to more clearly define the mechanism of action, optimum dose and scheduling, and clinical efficacy of levamisole in treating other malignancies.