Faculty Bibliography

Thalidomide is currently being evaluated for efficacy in alleviating some manifestations of HIV-1 infection. To determine whether thalidomide has any direct effects on HIV-1 infection, we investigated the effect of thalidomide and also of three structural analogs of thalidomide on HIV-1 replication in vitro in human monocyte-derived macrophages. The thalidomide analogs were previously shown to inhibit TNF-alpha production in vitro at much lower concentrations than thalidomide. In HIV-1-infected macrophages treated with thalidomide or thalidomide analogs, viral replication was reduced by 60 to 80% as determined by measuring viral RT activity in the culture supernatants. In all experiments the analogs inhibited HIV-1 replication more efficiently than did thalidomide. The drugs also reduced HIV-1 gag mRNA expression. Furthermore, the drugs caused a decrease in NF-kappaB-binding activity in nuclear extracts of HIV-1-infected macrophages. The role of NF-kappaB in the drug-induced inhibition of HIV-1 replication was confirmed using an NF-kappaB-defective mutant virus to infect macrophages

Following aerosol infection of mice with Mycobacterium tuberculosis, single mycobacteria or pairs of bacilli were observed within individual phagocytic vacuoles bound by tightly apposed vacuolar membranes. The virulent organism was not observed free in the cytoplasm of the parasitized cells or in the extracellular space of the lung granulomata. This study indicates that in vivo, virulent mycobacteria survive and probably replicate within a unique tight vacuole in the infected phagocyte within the lung

Host defense against mycobacterial infection requires the participation of monocytes and T cells. Both CD4+ and CD8+ T cells have been shown to be important in resistance to mycobacterial infection in vivo. The main contribution of CD4+ T cells to the protective antituberculosis response involves the production of Th1-type cytokines, including interleukin-2 (IL-2) and gamma interferon (IFN-gamma). CD8+ T cells have been considered to be responsible primarily for cytotoxicity mediated by toxic molecules, including perforin. CD8+ T cells may also elaborate Th1-type cytokines, such as IFN-gamma, in response to the infection. To elucidate the contribution of perforin-mediated target cell death to the control of mycobacterial infection in vivo, mice with a disruption in the perforin gene (P-/-) were infected with Mycobacterium bovis BCG or M. tuberculosis Erdman for 5 and 13 weeks, respectively. At 1, 3, 5, and 13 weeks postinfection, the number of viable mycobacteria in the lungs, spleens, and livers of mice were determined by CFU assay. The infected tissues were examined histologically, and cytokine mRNA levels in the spleens of these mice were determined. Similar studies were carried out in Fas receptor-defective (CBA/lpr(cg)) mice to evaluate the contribution of this alternative cytotoxic pathway to the control of mycobacterial infection. The absence of either perforin gene function or Fas receptor gene function did not modify the course of experimental mycobacterial infection in these mice. In addition, both P-/- and Fas receptor-defective mice appeared to have a compensatory activation of cytokine genes, even in the absence of the experimental infection. P-/- mice had a mean 3.4- to 5-fold increase in mRNA levels for IL-10, IL-12p35, IL-6, and IFN-gamma. Similarly, Fas receptor-defective mice had a mean 3- to 3.6-fold increase in mRNA levels for IFN-gamma, IL-12p35, and IL-10. Our results indicate that both perforin-mediated cytotoxicity and Fas-mediated cytotoxicity do not appear to be necessary for the early control of mycobacterial infection in vivo

SETTING: Experimental murine tuberculosis. OBJECTIVE: To evaluate the effect of cytokine modulation by thalidomide on the progression of the lung granulomatous response following aerosol tuberculosis infection in mice. DESIGN: Mice infected by the respiratory route with 200-500 viable Mycobacterium tuberculosis Erdman were treated with daily subcutaneous injections of thalidomide (30 mg/kg) or saline for 4 weeks. The bacillary load, granulomatous response and cytokine production in the lungs were evaluated. RESULTS: Aerosol M. tuberculosis infection resulted in a progressive granulomatous response in the lungs. At 28 days after infection, large granulomata with central necrosis and no apoptosis were observed. The infection induced high serum and lung cytokine mRNA levels. Thalidomide treatment resulted in a significant reduction in tumor necrosis factor-alpha, interleukin 6 (IL-6) and IL-10 protein levels (blood) and mRNA expression (lungs). IL-12 and interferon-gamma were unaffected. The lungs of thalidomide-treated mice had smaller granulomata with apoptotic cells and no necrosis. Thalidomide treatment did not change the bacillary load. CONCLUSION: Thalidomide immunomodulation reduces inflammatory cytokines and concomitant lung pathology following acute aerosol M. tuberculosis infection, without increasing the bacillary load

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) is thought to mediate both protective and detrimental manifestations of the inflammatory response. Recently, thalidomide (alpha-N-phthalimidoglutarimide) was shown to partially inhibit monocyte TNF alpha production (by 50-70%) both in vivo and in vitro. More efficient inhibition of TNF alpha may, however, be necessary to rescue the host from more acute and extensive toxicities of TNF alpha-mediated inflammation. MATERIALS AND METHODS: Three structural analogues of thalidomide were selected for study based on increased activity against TNF alpha production. The parent drug and the analogs were tested in vitro in human peripheral blood mononuclear cell cultures for their effects on lipopolysaccharide (LPS) induced cytokine protein and mRNA production using ELISAs and Northern blot hybridization. The in vitro effects of the drugs were then confirmed in vivo in a mouse model of LPS induced lethality. RESULTS: The new compounds (two esters and one amide) showed increased inhibition of TNF alpha production by LPS-stimulated human monocytes, relative to the parent drug thalidomide. The analogs and the parent drug enhanced the production of interleukin 10 (IL-10), but had little effect on IL-6 and IL-1 beta protein and mRNA production. When tested in vivo, the amide analog protected 80% of LPS-treated mice against death from endotoxin induced shock. CONCLUSIONS: Analogs of thalidomide designed to better inhibit TNF alpha production in vitro have correspondingly greater efficacy in vivo. These finding may have therapeutic implication for the treatment of human diseases characterized by acute and extensive TNF alpha production such as tuberculous meningitis or toxic shock

The immune responses to Mycobacterium leprae and other mycobacterial antigens were studied in 11 leprosy patients with concurrent human immunodeficiency virus type 1 (HIV-1) infection. Three patients manifested borderline lepromatous leprosy, and eight patients had borderline tuberculoid (BT) leprosy. Despite the low CD4+ T-cell count in the peripheral blood, no histologic or phenotypic change in the cellular infiltrate in either the lepromatous or tuberculoid lesions was observed when compared with HIV-1-negative patients. Lepromatous lesions contained heavily parasitized macrophages and few CD8+ T cells. Lesions from the patients with BT leprosy showed extensive CD4+ T-cell infiltration despite a significant reduction in CD4+ T-cell counts in the peripheral blood. No acid-fast bacilli were detected in the tuberculoid lesions. HIV-1 infection did not alter the lack of response in lepromatous leprosy to M. leprae antigens either in vitro or in vivo. In contrast, the skin test response to M. leprae antigens as well as the in vitro lymphoproliferative responses to mycobacterial antigens that are usually seen in patients with tuberculoid leprosy were abrogated in the BT HIV-1+ patients. However, production of gamma interferon in response to the same stimuli was preserved in most of the patients. Analysis of cytokine gene expression showed activation of additional cytokine genes in the unstimulated peripheral blood cells of patients with both leprosy and HIV-1 infections as compared with cells from patients with leprosy alone. These results suggest that granuloma formation in leprosy can be independent of the impaired CD4+ T-cell response of the HIV-1 infection. Furthermore, in HIV-1+ individuals with M. leprae infection, activation of cytokine genes is observed even when the circulating CD4+ T-cell count is significantly reduced

In this study, we evaluated the activity of peripheral blood mononuclear cells (PBMC), isolated from treated and untreated lepromatous leprosy patients, from lepromatous leprosy patients during and after reactional episodes (erythema nodosum leprosum (ENL) and reversal reaction (RR)), and from normal healthy individuals. We determined reactive oxygen intermediate (ROI) production, procoagulant activity (PCA) and HLA-DR antigen expression of monocytes, besides lymphoproliferation, both in the presence and absence of various stimulatory agents. Phorbol myristate acetate (PMA) stimulated ROI production by monocytes from all the groups studied, with patients during reactional episodes (ENL and RR) showing a significantly higher response (p < 0.009 and p < 0.00001). Irradiated Mycobacterium leprae, although having little effect when added alone, strongly suppressed PMA-stimulated ROI production. Muramyl dipeptide (MDP) had no influence on either basal or on PMA-induced ROI production. Basal monocyte PCA, as well as M. leprae or concanavalin A (ConA)-induced monocyte PCA was comparable in monocytes from all the groups studied. ConA was able to induce mitogenic activity in mononuclear cells isolated from all the groups studied. M. leprae, although stimulatory for normal individuals, did not induce lymphoproliferation in lepromatous leprosy patients, except for cells from patients during RR, which responded equally to M. leprae and to ConA. The absence of M. leprae-induced lymphoproliferation in lepromatous leprosy patients is not caused by the lack of basal HLA-DR expression, as PBMC from all individuals studied showed the same level of this antigen. Our results suggest an increase of spontaneous or PMA-induced monocyte activity, as detected by ROI production, during the reactional episode; addition of M. leprae suppressed this response. The increase in monocyte activity could be correlated with the increase of lymphoproliferation response to M. leprae during RR, but not during ENL. The importance of a possible immune suppressive action of M. leprae is discussed

Thalidomide, a selective inhibitor of tumor necrosis factor alpha (TNF-alpha) synthesis, suppresses the activation of latent human immunodeficiency virus type 1 (HIV-1) in a monocytoid (U1) line. The inhibition is dose dependent and occurs after exposure of the cells to recombinant TNF-alpha, phorbol myristate acetate, lipopolysaccharide, and other cytokine combinations. Associated with HIV-1 inhibition is a reduction in agonist-induced TNF-alpha protein and mRNA production. Thalidomide inhibition of virus replication in the phorbol myristate acetate- and recombinant TNF-alpha-stimulated T-cell line ACH-2 is not observed. The presence of thalidomide also inhibits the activation of virus in the peripheral blood mononuclear cells of 16 out of 17 patients with advanced HIV-1 infection and AIDS. These results suggest the use of thalidomide in a clinical setting to inhibit both virus replication and the TNF-alpha-induced systemic toxicity of HIV-1 and opportunistic infections

We have examined the mechanism of thalidomide inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production and found that the drug enhances the degradation of TNF-alpha mRNA. Thus, the half-life of the molecule was reduced from approximately 30 to approximately 17 min in the presence of 50 micrograms/ml of thalidomide. Inhibition of TNF-alpha production was selective, as other LPS-induced monocyte cytokines were unaffected. Pentoxifylline and dexamethasone, two other inhibitors of TNF-alpha production, are known to exert their effects by means of different mechanisms, suggesting that the three agents inhibit TNF-alpha synthesis at distinct points of the cytokine biosynthetic pathway. These observations provide an explanation for the synergistic effects of these drugs. The selective inhibition of TNF-alpha production makes thalidomide an ideal candidate for the treatment of inflammatory conditions where TNF-alpha-induced toxicities are observed and where immunity must remain intact