In vivo development of fluconazole resistance in serial Cryptococcus gattii isolates from a cat.

Elevated fluconazole minimum inhibitory concentrations (MICs) are more frequently observed in Cryptococcus gattii compared to C. neoformans isolates; however, the development of in vivo resistance and the molecular mechanisms responsible have not been reported for this species. We report a case of Cryptococcus gattii (molecular type VGIII) that developed reduced susceptibility to fluconazole during therapy and delineate the molecular mechanisms responsible. Multilocus sequence typing and quantitative DNA analysis of the pre- and post-treatment isolates was performed using well-characterized methods. Pre- and post-treatment clinical isolates were confirmed isogenic, and no differences in ERG11 or PDR11 sequences were found. qPCR found an overexpression of ERG11 and the efflux pump PDR11 in the resistant isolate compared to the isolate collected prior to initiation of antifungal therapy. Reversion to wild-type susceptibility was observed when maintained in antifungal-free media confirming the in vivo development of heteroresistance. The in vivo development of heteroresistance to fluconazole in our patient with C. gattii is secondary to overexpression of the efflux pump PDR11 and the drug target ERG11. Additional work in other clinical isolates with elevated fluconazole MICs is warranted to evaluate the frequency of heteroresistance versus point mutations as a cause of resistance.

In vitro interactions between amphotericin B and hydrocortisone: potential implications for intrathecal therapy.

Fungal meningitis remains a severe and often lethal infection requiring aggressive antifungal therapy and in refractory cases the use of intrathecal amphotericin B (AmB). Administration of amphotericin B by this method may result in clinically apparent adverse reactions such as paresthesias, radiculitis, or myelopathy. Coadministration of hydrocortisone is therefore often given in an attempt to avoid these effects; however, the potential consequences of this approach on fungal growth or on drug synergy/antagonism had not previously been assessed. We used the checkerboard titration broth microdilution method to analyze interactions by fractional inhibitory concentration indices (FICIs). The combination of amphotericin B and hydrocortisone resulted in synergy or indifference against all isolates (Candida, Cryptococcus, and Coccidioides) during in vitro testing at low concentrations. Antagonism was observed using higher hydrocortisone concentrations (those not observed in vivo) suggesting possible steric hindrance or binding to AmB may occur at doses unlikely to be present during clinical care. Concurrent hydrocortisone and AmB administration should not be avoided due to in vitro antagonism concerns.

Increased natural killer T-like cells are a major source of pro-inflammatory cytokines and granzymes in lung transplant recipients.

BACKGROUND AND OBJECTIVE: Natural killer T (NKT)-like cells are a small but significant population of T lymphocytes; however, their role in lung transplant and the effect of current immunosuppressive agents on their function is largely unknown. We have previously shown lung transplant rejection was associated with an increase in peripheral blood T cell γ-interferon (IFN-γ), tumour necrosis factor-α (TNF-α) and granzyme B. NKT-like cells are a source of these pro-inflammatory mediators and as such may be involved in lung transplant pathology. METHODS: We analysed NKT-like cell numbers and cytokine and granzyme profiles in peripheral blood from a group of stable lung transplant patients and control subjects using multiparameter flow cytometry. RESULTS: There was a significant increase in NKT-like cells in transplant patients compared with control subjects (6.8 ± 4.9 vs 0.8 ± 0.2% lymphocytes respectively). There was an increase in the numbers of NKT-like cells producing IFN-γ, TNF-α, IL-2 IL-17, granzyme and perforin in transplant patients compared with controls. Immunosuppressant drugs were less effective at inhibiting IFN-γ and TNF-α production by T and NKT-like cells than NK cells in vitro. CONCLUSIONS: Current therapeutics is inadequate at suppressing NKT-like cell numbers and their production of pro-inflammatory mediators known to be associated with graft rejection. Alternative therapies that specifically target NKT-like cells may improve patient morbidity.

Garlic compounds selectively kill childhood pre-B acute lymphoblastic leukemia cells in vitro without reducing T-cell function: Potential therapeutic use in the treatment of ALL.

Drugs used for remission induction therapy for childhood precursor-B acute lymphoblastic leukemia (ALL) are nonselective for malignant cells. Several garlic compounds have been shown to induce apoptosis of cancer cells and to alter lymphocyte function. To investigate the effect of garlic on the apoptosis of ALL cells and lymphocyte immune function, cells from newly diagnosed childhood ALL patients were cultured with several commonly used chemotherapeutic agents and several garlic compounds. Apoptosis, lymphocyte proliferation and T-cell cytokine production were determined using multiparameter flow cytometry. At concentrations of garlic compounds that did not result in significant increases in Annexin V and 7-AAD staining of normal lymphocytes, there was a significant increase in apoptosis of ALL cells with no alteration of T-cell proliferation as determined by CD25/CD69 upregulation or interferongamma, interleukin-2 or tumor necrosis factor-alpha intracellular cytokine production. In contrast, the presence of chemotherapeutic agents resulted in nonselective increases in both lymphocyte and ALL apoptosis and a decrease in T-cell proliferation and cytokine production. In conclusion, we show selective apoptosis of malignant cells by garlic compounds that do not alter T-cell immune function and indicate the potential therapeutic benefit of garlic compounds in the treatment of childhood ALL.

Allium sativum (garlic) suppresses leukocyte inflammatory cytokine production in vitro: potential therapeutic use in the treatment of inflammatory bowel disease.

BACKGROUND: Cytokines involved in inflammatory bowel disease (IBD) direct a predominantly cell-mediated T- helper-1 (Th1) immune response. The nonspecific anti-inflammatory treatment being used in the management of patients with IBD has not changed much since the 1970s and new therapeutic agents are keenly sought. Several compounds isolated from Allium sativum (garlic) modulate leukocyte cell proliferation and cytokine production. METHODS: To investigate the possible therapeutic effects of garlic in the treatment of patients with IBD, whole blood and peripheral blood mononuclear cells (PBMCs) were stimulated in the presence of various concentrations of garlic extract and the effect on leukocyte cytokine production was determined in vitro using multiparameter flow cytometry. RESULTS: Monocyte interleukin (IL)-12 production was inhibited significantly in the presence of low concentrations of garlic extract (>or=0.1 microg/ml total protein). Monocyte IL-10 production increased significantly and monocyte tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, IL-6, IL-8, T-cell interferon-gamma (IFN-gamma), IL-2, and TNF-alpha decreased significantly in the presence of >or=10 microg/ml garlic extract. Twenty to fifty percent of the immunomodulatory activity of garlic extract on cytokine production was acid labile. The inhibitory activity of methylprednisolone, a commonly used anti-inflammatory in IBD, with garlic on leukocyte cytokine production was additive. CONCLUSIONS: By inhibiting Th1 and inflammatory cytokines while upregulating IL-10 production, treatment with garlic extract may help to resolve inflammation associated with IBD. An in vivo animal model study needs to be undertaken to determine the significance of these in vitro findings.