Oral antimycobacterial therapy in chronic cutaneous sarcoidosis: a randomized, single-masked, placebo-controlled study.

IMPORTANCE: Sarcoidosis is a chronic granulomatous disease for which there are limited therapeutic options. This is the first randomized, placebo-controlled study to demonstrate that antimycobacterial therapy reduces lesion diameter and disease severity among patients with chronic cutaneous sarcoidosis. OBJECTIVE: To evaluate the safety and efficacy of once-daily antimycobacterial therapy on the resolution of chronic cutaneous sarcoidosis lesions. DESIGN AND PARTICIPANTS: A randomized, placebo-controlled, single-masked trial on 30 patients with symptomatic chronic cutaneous sarcoidosis lesions deemed to require therapeutic intervention. SETTING: A tertiary referral dermatology center in Nashville, Tennessee. INTERVENTIONS: Participants were randomized to receive either the oral concomitant levofloxacin, ethambutol, azithromycin, and rifampin (CLEAR) regimen or a comparative placebo regimen for 8 weeks with a 180-day follow-up. MAIN OUTCOMES AND MEASURES: Participants were monitored for absolute change in lesion diameter and decrease in granuloma burden, if present, on completion of therapy. OBSERVATIONS: In the intention-to-treat analysis, the CLEAR-treated group had a mean (SD) decrease in lesion diameter of -8.4 (14.0) mm compared with an increase of 0.07 (3.2) mm in the placebo-treated group (P = .05). The CLEAR group had a significant reduction in granuloma burden and experienced a mean (SD) decline of -2.9 (2.5) mm in lesion severity compared with a decline of -0.6 (2.1) mm in the placebo group (P = .02). CONCLUSIONS AND RELEVANCE: Antimycobacterial therapy may result in significant reductions in chronic cutaneous sarcoidosis lesion diameter compared with placebo. These observed reductions, associated with a clinically significant improvement in symptoms, were present at the 180-day follow-up period. Transcriptome analysis of sarcoidosis CD4+ T cells revealed reversal of pathways associated with disease severity and enhanced T-cell function following T-cell receptor stimulation. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01074554.

Reversal of global CD4+ subset dysfunction is associated with spontaneous clinical resolution of pulmonary sarcoidosis.

Sarcoidosis pathogenesis is characterized by peripheral anergy and an exaggerated, pulmonary CD4(+) Th1 response. In this study, we demonstrate that CD4(+) anergic responses to polyclonal TCR stimulation are present peripherally and within the lungs of sarcoid patients. Consistent with prior observations, spontaneous release of IL-2 was noted in sarcoidosis bronchoalveolar lavage CD4(+) T cells. However, in contrast to spontaneous hyperactive responses reported previously, the cells displayed anergic responses to polyclonal TCR stimulation. The anergic responses correlated with diminished expression of the Src kinase Lck, protein kinase C-θ, and NF-κB, key mediators of IL-2 transcription. Although T regulatory (Treg) cells were increased in sarcoid patients, Treg depletion from the CD4(+) T cell population of sarcoidosis patients did not rescue IL-2 and IFN-γ production, whereas restoration of the IL-2 signaling cascade, via protein kinase C-θ overexpression, did. Furthermore, sarcoidosis Treg cells displayed poor suppressive capacity indicating that T cell dysfunction was a global CD4(+) manifestation. Analyses of patients with spontaneous clinical resolution revealed that restoration of CD4(+) Th1 and Treg cell function was associated with resolution. Conversely, disease progression exhibited decreased Th1 cytokine secretion and proliferative capacity, and reduced Lck expression. These findings implicate normalized CD4(+) T cell function as a potential therapeutic target for sarcoidosis resolution.

Sarcoidosis Th17 cells are ESAT-6 antigen specific but demonstrate reduced IFN-γ expression.

RATIONALE: Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells. METHODS: Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation. RESULTS: Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p = 0.03 and p = 0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (p < 0.001 and p = 0.03, respectively). After polyclonal stimulation, Th-17 cells from sarcoidosis patients produced less IFN-γ than healthy controls. CONCLUSIONS: Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis.

Cholesterol depletion inactivates XMRV and leads to viral envelope protein release from virions: evidence for role of cholesterol in XMRV infection.

Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-ß-cyclodextrin (2OHpßCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpßCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.

Ziram activates mitogen-activated protein kinases and decreases cytolytic protein levels in human natural killer cells.

Human natural killer (NK) cells are central in immune defense with their ability to lyse tumor cells and virally infected cells. Tumor formation and viral infection may increase if NK cytotoxic function is disrupted. Ziram (zinc dithiocarbamate) is used as an accelerating agent in the production of latex and to protect various fruits and vegetables from fungal infection. Previously, we have shown that exposure to ziram inhibits NK lytic function. Butyltin environmental contaminants, which also inhibit NK lytic function, cause rapid activations of mitogen-activated protein kinases (MAPKs) and decreases in expression of the cytolytic proteins granzyme B and perforin (after 24 h) in exposed NK cells. MAPKs are important regulators of the lytic response of NK cells, and spurious activation of these enzymes by contaminants would leave the NK cells unable to respond to appropriate targets. This study examined the effects of ziram exposures on MAPKs (p44/42, p38, and c-jun-N-terminal kinase) and on levels of cytolytic proteins. Ten-minute to 6-h exposures of NK cells to ziram caused activation of MAPKs, p44/42, and p38. Exposure to ziram for 24 h caused a decrease in granzyme B and perforin levels. MAPK inhibitors were able to prevent these ziram-induced decreases in granzyme B and perforin. These results suggest that ziram-induced MAPK activation is at least in part responsible for decreased cytolytic function in ziram-exposed NK cells. Furthermore, the results indicate that these changes are in common with other environmental contaminants that have been shown to decrease NK lytic function.

Effects of ziram on tumor-cell-binding capacity, cell-surface marker expression, and ATP levels of human natural killer cells.

Human natural killer (NK) cells are central in immune defense against tumor and virally infected cells. Ziram is used as an accelerating agent in latex production and as an agricultural fungicide. Previous studies showed that continuous exposure to ziram inhibits NK lytic function. Additionally, they showed that a brief (1 h) exposure to ziram caused persistent loss of lytic function. This study examined whether decreases in lytic function were accompanied by decreases in the target-binding function of NK cells and found that some, but not all, exposures to ziram caused significant decreases in binding function. Ziram exposures that caused a loss of binding function were examined for effects on expression of key NK cell-surface proteins needed for binding to targets. Exposure to 2 microM ziram for 1 h followed by 24 or 48 h in ziram-free media decreased CD16 expression, but no other exposures caused decreases in cell-surface proteins. As decreases in adenosine triphosphate (ATP) could be in part responsible for loss of lytic function, the effect of ziram exposures on ATP levels of NK cells were examined. Certain ziram exposures decreased ATP levels in NK cells, but a decrease in ATP was not necessarily associated with a decrease in lytic function. The results indicate that ziram-induced losses of lytic function cannot be fully explained by alteration in binding, cell-surface protein expression, or ATP levels.

Persistent inhibition of human natural killer cell function by ziram and pentachlorophenol.

Ziram is a currently used agricultural fungicide. It is also used as an additive in the production of latex gloves. Because of these uses, there is a potential for human exposure to this compound. Pentachlorophenol (PCP) has been used as an insecticide, fungicide, disinfectant, and ingredient in antifouling paints. Currently, it is used as a wood preservative for power-line poles and fence posts. Measurable levels of PCP have been detected in human blood and urine. In previous studies we demonstrated that both these compounds could cause very significant inhibition of the tumor-killing function of human natural killer (NK) cells. NK lymphocytes play a central role in immune defense against viral infection and the formation of primary tumors. So interference with their function could increase the risk of tumor development. In the present study we examined the effects of exposure to ziram or PCP of brief duration (1 h) on the ability of NK cells to destroy tumor cells. NK cells were exposed to either ziram (5-0.5 microM) or PCP (10-5 microM) for 1 h followed by 0 h, 24 h, 48 h, or 6 days in compound-free media and then were tested for the ability to lyse as well as to bind tumor cells. A 1-h exposure to as little as 2.5 microM ziram decreased the ability of NK cells to lyse target tumor cells, which persisted up to 6 days following exposure. The loss of lytic function for from 24 h to 6 days following exposure was accompanied by a comparable loss of NK capacity to bind tumor cells. Exposure to 10 microM PCP for 1 h caused a progressive loss (greater than 80%) of lytic function within 6 days of exposure. In contrast to ziram, PCP exposure caused no accompanying loss of binding function.