Felipe Sandoval

Thalidomide increases the synthesis of IL-2 in cultures of human mononuclear cells stimulated with Concanavalin-A, staphylococcal enterotoxin A, and purified protein derivative

Thalidomide significantly increases the quantity of extracellular IL-2 in cultures of human mononuclear cells stimulated with mitogens or antigen. Cells from 7 donors exposed for 2 h to 4.0 micrograms/ml of thalidomide and stimulated for 16-18 h with 20 micrograms/ml of Concanavalin-A (Con-A) averaged producing 187 +/- 49% more IL-2 than cells stimulated with Con-A alone. In similar experimental procedures and comparisons the pg/ml of IL-2 secreted by thalidomide-treated cells from five donors stimulated with 50 ng/ml of Staphylococcal enterotoxin A (SEA) increased by 159 +/- 32%, and the pg/ml of IL-2 secreted by thalidomide-treated cells from 2 donors stimulated with 5.0 micrograms/ml of purified protein derivative of Mycobacterium tuberculosis increased by 120 +/- 4%. Thalidomide also significantly increases the quantity of intracellular IL-2 in cells stimulated with mitogens. Cells exposed to thalidomide and stimulated with Con-A had an increase in intracellular IL-2 of 130% after 8 h and 157% after 12 h in culture; cells stimulated with SEA had an increase in intracellular IL-2 of 120% after 8 h and 182% after 12 h in culture. Thalidomide did not alter the percent of lymphocytes expressing the alpha-chain of IL-2 receptor, nor did it significantly increase incorporation of [3H]thymidine by cells.

Thalidomide Can Be Either Agonistic or Antagonistic to LPS Evoked Synthesis of Tnf-α by Mononuclear Cells

The effect if thalidomide on tumor necrosis factor alpha (TNF-alpha) produced in vitro by lipopolysaccharide (LPS) stimulated human cells was investigated. In cultures of LPS stimulated human mononuclear cells enriched for adherent cells and in cultures of LPS stimulated human monocytes of the cell line-THP-1, thalidomide enhanced the synthesis of TNF-alpha. When cultures of unfractionated peripheral blood mononuclear cells were stimulated with LPS, thalidomide decreased the synthesis of TNF-alpha. Depending on the type of cells stimulated with LPS in vitro, thalidomide, at concentrations achieved in vivo, can either enhance or suppress the synthesis of TNF-alpha.

Hydrolysis of thalidomide abrogates its ability to enhance mononuclear cell synthesis of IL-2 as well as its ability to suppress the synthesis of TNF-α

Thalidomide is effective in the treatment of inflammatory conditions like erythema nodosum leprosum in leprosy patients, and aphthous ulcers in AIDS patients. Its mechanism of action is uncertain and reports of its effect on the synthesis of inflammatory cytokines such as IL-2 and TNF-alpha are contradictory. As thalidomide is labile to spontaneous hydrolysis at pH 7.4, studies were carried out to explore the effects of deliberate hydrolysis or the ability of thalidomide to modulated cytokine production by human mononuclear cells stimulated in vitro with Staphylococcal enterotoxin A (SEA)(IL-2) or lipopolysaccharide from Salmonella minnesota (LPS)(TNF-alpha). Unhydrolyzed thalidomide at 4.0 micrograms/ml consistently enhanced the synthesis of IL-2 in SEA-stimulated cells, and suppressed the synthesis of TNF-alpha in LPS-stimulated cells; whereas, hydrolyzed thalidomide had no enhancing effect on SEA stimulated-cell synthesis of IL-2 or suppressive effect on LPS stimulated-cell synthesis of TNF-alpha. These findings demonstrate that thalidomides ability in vitro to enhance IL-2 and to suppress TNF-alpha in stimulated cells is dependent on the intact molecule and underscore the necessity to employ thalidomide under appropriate physicochemical conditions.

Immunomodulatory assays to study structure-activity relationships of thalidomide.

Thalidomide, which has a long history of tragedy because of its ability to cause severe birth defects, is very effective in alleviating erythema nodosum leprosum in leprosy patients and aphthous ulcers in AIDS patients. The causes of these inflammatory diseases and the mechanism by which thalidomide diminishes them are unknown. It has been suggested that modulation of the immune response plays an important role. We found that thalidomide exerts immunomodulatory activity in three bioassays. It suppresses an IgM plaque forming cell response in mice injected with sheep erythrocytes: it inhibits TNF-alpha production by LPS stimulated human mononuclear cells: and it enhances IL-2 production by Con-A stimulated human mononuclear cells. We employed these bioassays to compare the activity of 15 analogs of thalidomide with thalidomide itself. Eight of the compounds were derivatives of the glutarimide moiety of thalidomide and the others were phthalimide or derivatives of the phthalimide moiety of thalidomide. N-hydroxyphthalimide, a simple derivative of phthalimide, was more effective than thalidomide and was also the most effective of the compounds assayed in suppressing the IgM plaque and TNF-alpha responses, but it did not enhance the IL-2 response, instead, it significantly suppressed it.

Gene Expression Profiling Specifies Chemokine, Mitochondrial and Lipid Metabolism Signatures in Leprosy

Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.

Thalidomide Suppressed IL-1β While Enhancing TNF-α and IL-10, When Cells in Whole Blood were Stimulated with Lipopolysaccharide

Thalidomide is used to treat erythema nodosum leprosum (ENL). The events that precipitate this inflammatory reaction, which may occur in multibacillary leprosy patients, and the mechanism by which thalidomide arrest ENL, are not known. Thalidomides ability to inhibit tumor necrosis factor alpha (TNF-α) in vitro has been proposed as a partial explanation of its effective treatment of ENL. In in vitro assays, thalidomide can enhance or suppress TNF-α. This is dependent on the stimulant used to evoke TNF-α; the procedure used to isolate the mononuclear cells from blood, and the predominant mononuclear cell type in the culture. To avoid artifacts that may occur during isolation of mononuclear cells from blood, we stimulated normal human blood with LPS and evaluated the effect of thalidomide and dexamethasone on TNF-α, and other inflammatory cytokines and biomarkers. Thalidomide suppressed interleukin 1 β (IL-1β) (p = 0.007), and it enhanced TNF-α (p = 0.007) and interleukin 10 (IL-10) (p = 0.031). Dexamethasone enhanced IL-10 (p = 0.013) and suppressed IL-1β, TNF-α, interleukin 6 (IL-6), and interleukin 8 (IL-8) (p = 0.013). The two drugs did not suppress: C-reactive protein (CRP), Ig-superfamily cell-adhesion molecule 1 (ICAM 1), tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor receptor 2 (TNFR2), or amyloid A. In vitro and in vivo evidence is accumulating that TNF-α is not the primary cytokine targeted by thalidomide in ENL and other inflammatory conditions.

Stabilization of Red Blood Cell Membranes by Thalidomide In Vitro

The anti‐inflammatory effect of thalidomide has been well established. The mechanism of this anti‐inflammatory action is still not completely understood. Certain drugs exert their anti‐inflammatory action by stabilizing the membranes of polymorphonuclear neutrophils (PMN) thereby reducing the production of reactive oxygen intermediates. We evaluated the effect of thalidomide on cell membranes by using red blood cells (RBC), PMN and the monocyte‐like cell line THP‐1. Osmotic fragility of RBC showed that in vitro, thalidomide stabilized the membrane of RBC from plasma free blood; whereas, it did not affect RBCs from whole blood. Red blood cells taken from subjects before and after ingestion of thalidomide were not affected after exposure to different concentrations of hypotonic NaCl solution. Thalidomide did not affect the membrane stability of PMNs as well as THP‐1 in a significant manner. These data suggest that the anti‐inflammatory mechanism of thalidomide is not related to events associated with the oxidative burst of PMNs or monocytes.

Drug Resistance in Patients With Leprosy in the United States

Molecular drug susceptibility testing was performed on 39 US patients with leprosy. Of these, 2 had dapsone-resistant Mycobacterium leprae and 1 of these patients also had rifampin-resistant M. leprae. Even though antileprosy drug resistance occurs in this leprosy population, resistance does not appear to be a major problem.

Thalidomide delayed the ability of 4T1 cells to amass into tumors in Balb/c mice.

Thalidomide (Thal) can suppress the growth of established, as well as explanted tumors in mice. We wanted to determine if it could suppress the ability of tumor cells to assemble and establish a primary tumor at the injection site. Using the mouse 4T1 mammary tumor model, we fed Thal to mice for 4 days, then injected 105 4T1 cells into the interscapular region of Balb/c mice. After 20 days on treatment with Thal, all seven control mice, fed with meal had tumors ranging from 3 to 93 mm3 (median 20). Two of the eight mice fed with meal + Thal had no tumors, and the remaining mice had tumors ranging from 2 to 22 mm3 (median 5). The median volume of the tumors in the control group was significantly more than that of mice treated with Thal (p = 0.03, Mann–Whitney test). In vitro treatment of the 4T1cells with Thal did not inhibit their ability to proliferate, to adhere to plastic, or to bind to Concanavalin-A. Thal caused a marked reduction in the ability of the 4T1 cells to assemble into palpable tumors.