Azaphilones from an Acid Mine Extremophile Strain of a Pleurostomophora sp.

An extremophilic fungus identified as a Pleurostomophora sp. was isolated from the Berkeley Pit, an acid mine waste lake. When grown in liquid culture, the fungus produced berkchaetoazaphilones A-C (1, 2, and 5), the red pigment berkchaetorubramine (6), and the known compound 4-(hydroxymethyl)quinoline. These compounds were evaluated as inhibitors of matrix metalloproteinase-3, caspase-1, and proinflammatory cytokine production in induced THP-1 cells. Berkchaetoazaphilone B (2) inhibited IL-1ß, TNFα, and IL-6 production in the induced inflammasome assay and was cytotoxic toward human retinoblastoma cell line Y79 (IC50 = 1.1 µM), leukemia cell lines CCRF-CEM and SR, and the melanoma cell line LOX IMVI (IC50 = 10 µM).

IL-1R signalling is critical for regulation of multi-walled carbon nanotubes-induced acute lung inflammation in C57Bl/6 mice.

Exposure to certain engineered nanomaterials has been associated with pathological changes in animal models raising concerns about potential human health effects. MWCNT have been reported to activate the NLRP3 inflammasome in vitro, correlating with lung inflammation and pathology, in vivo. In this study, we investigated the role of IL-1 signalling in pulmonary inflammatory responses in WT and IL-1R-/- mice after exposure to MWCNT. The results suggest that MWCNT were effective in inducing acute pulmonary inflammation. Additionally, WT mice demonstrated significant increased airway resistance 24 h post exposure to MWCNT, which was also blocked in the IL-1R-/- mice. In contrast, by 28 days post exposure to MWCNT, the inflammatory response that was initially absent in IL-1R-/- mice was elevated in comparison to the WT mice. These data suggest that IL-1R signalling plays a crucial role in the regulation of MWCNT-induced pulmonary inflammation.

Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium.

BACKGROUND: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity. OBJECTIVES: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability. METHODS: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1ß (IL-1ß) release] using only THP-1 cells. RESULTS: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 µg/mL, but did not induce IL-1ß. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1ß production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1ß production in THP-1 cells, with the original MWCNT producing the most IL-1ß. CONCLUSIONS: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

IL-33 mediates multi-walled carbon nanotube (MWCNT)-induced airway hyper-reactivity via the mobilization of innate helper cells in the lung.

Allergic asthma is a chronic inflammatory disorder of the airway associated with bronchial obstruction, airway hyper-reactivity (AHR), and mucus production. The epithelium may direct and propagate asthmatic-like responses. Central to this theory is the observation that viruses, air pollution, and allergens promote epithelial damage and trigger the generation of IL-25, IL-33, and TSLP via innate pathways such as TLRs and purinergic receptors. Similarly, engineered nanomaterials promote a Th2-associated pathophysiology. In this study, we tested the hypothesis that instillation of multi-walled carbon nanotubes (MWCNT) impair pulmonary function in C57Bl/6 mice due to the development of IL-33-dependent Th2-associated inflammation. MWCNT exposure resulted in elevated levels of IL-33 in the lavage fluid (likely originating from airway epithelial cells), enhanced AHR, eosinophil recruitment, and production of Th2-associated cytokines and chemokines. Moreover, these events were dependent on IL-13 signaling and the IL-33/ST2 axis, but independent of T and B cells. Finally, MWCNT exposure resulted in the recruitment of innate lymphoid cells. Collectively, our data suggest that MWCNT induce epithelial damage that results in release of IL-33, which in turn promotes innate lymphoid cell recruitment and the development of IL-13-dependent inflammatory response.

Caspase-1 inhibitors from an extremophilic fungus that target specific leukemia cell lines.

Berkeley Pit Lake, Butte, Montana, is a 540 m deep abandoned open-pit copper mine filled with over 140 billion liters of acidic, metal-sulfate-contaminated water. This harsh environment has yielded several microorganisms that produce interesting biologically active compounds. Several polyketide metabolites including the new berkazaphilones A (1) and B (2) and octadienoic acid derivatives berkedienoic acid (13) and berkedienolactone (15), as well as previously reported azaphilone 4, vermistatin (6), dihydrovermistatin (7), penisimplicissin (8), aldehyde 9, and methylparaconic acid (11), were isolated from a culture broth of Penicillium rubrum taken from a depth of 270 m. The structures of these compounds were deduced by interpretation of spectroscopic data. The compounds were isolated either for their inhibition of the signal transduction enzyme caspase-1 or because of their structural similarity to these inhibitors. Selected compounds were further evaluated for their ability to inhibit interleukin-1ß production by inflammasomes in induced THP-1 cells. Berkazaphilones B (2) and C (4) and vermistatin analogue penisimplicissin (8) exhibited selective activity against leukemia cancer cell lines in the National Cancer Institute 60 human cell line assay.

Caspase-1 and -3 inhibiting drimane sesquiterpenoids from the extremophilic fungus Penicillium solitum.

Two new drimane sesquiterpene lactones and one new tricarboxylic acid derivative were isolated from the Berkeley Pit extremophilic fungus Penicillium solitum. The structures of these compounds were deduced by spectroscopic analysis. Berkedrimanes A and B inhibited the signal transduction enzymes caspase-1 and caspase-3 and mitigated the production of interleukin 1-ß in the induced THP-1 (pro-monocytic leukemia cell line) assay.

Berkeleyones and related meroterpenes from a deep water acid mine waste fungus that inhibit the production of interleukin 1-ß from induced inflammasomes.

The Berkeley Pit, an acid mine waste lake, is a source of extremophilic microorganisms that produce interesting bioactive compounds. We have previously reported the isolation of berkeleydione (1), berkeleytrione (2), the berkeleyacetals, and the berkeleyamides from the Pit Lake fungus Penicillium rubrum. In this paper we report the isolation and characterization of berkeleyones A-C (4, 5, and 7) as well as previously described preaustinoid A (3) and A1(6) from this same fungus. These compounds were evaluated as inhibitors of the signaling enzyme caspase-1 and as potential inhibitors of interleukin 1-ß production by inflammasomes in induced THP-1 cell line assays.

Natural Foxp3(+) regulatory T cells inhibit Th2 polarization but are biased toward suppression of Th17-driven lung inflammation.

nTregs prevent autoimmunity and modulate immune and inflammatory responses to foreign antigens. CD4(+)Foxp3(+) nTregs from DO11.10 mice were expanded ex vivo, and their effectiveness in suppressing the development of lung inflammatory responses, elicited by differentiated CD4(+) T cells following antigen inhalation, was examined. Effector DO11.10 CD4(+) Th2 cells, when adoptively transferred into BALB/c mice that subsequently inhaled OVA, elicited a pronounced pulmonary, eosinophilic inflammation. Surprisingly, the cotransfer of expanded nTregs failed to suppress the Th2-mediated airway inflammation. Nevertheless, expanded OVA-specific CD4(+)Foxp3(+) nTregs were highly effective at inhibiting the polarization of naïve CD4(+) T cells into a Th2 phenotype. This suppression was reversed by an antibody to GITR but was not affected by the presence of the soluble OX40L. Further analysis revealed that although nTregs also failed to inhibit the lung neutrophilic inflammation induced by effector CD4(+) Th1 cells, they markedly suppressed pulmonary inflammation elicited by CD4(+) Th17 cells but not AHR. The suppression of the Th17-mediated response was evident from a striking reduction in the proportion of OVA-specific T cells expressing IL-17 and the numbers of neutrophils present in the airways of Th17 recipient mice. Collectively, these results demonstrate that expanded nTregs clearly limit the Th2 polarization process and that Th17-mediated inflammatory responses are particularly prone to the immunoregulatory properties of nTregs. These findings thus indicate that expanded nTregs are restrictive in their ability to suppress airway inflammatory processes and AHR.

Antigen-specific Treg regulate Th17-mediated lung neutrophilic inflammation, B-cell recruitment and polymeric IgA and IgM levels in the airways.

Th17 cells play key roles in mediating autoimmunity, inflammation and mucosal host defense against pathogens. To determine whether naturally occurring Treg (nTreg) limit Th17-mediated pulmonary inflammation, OVA-specific CD4+ Th17 cells and expanded CD4+CD25+Foxp3+ nTreg were cotransferred into BALB/c mice that were then exposed to OVA aerosols. Th17 cells, when transferred alone, accumulated in the lungs and posterior mediastinal LN and evoked a pronounced airway hyperreactivity and neutrophilic inflammation, characterized by B-cell recruitment and elevated IgA and IgM levels. Cotransfer of antigen-specific nTreg markedly reduced the Th17-induced pulmonary inflammation and associated neutrophilia, B-cell influx and polymeric Ig levels in the airways, but did not inhibit airway hyperreactivity. Moreover, the regulation appeared restricted to the site of mucosal inflammation, since transfer of nTreg did not affect the Th17 response developing in the lung draining LN, as evidenced by unaltered levels of IL-17 production and low numbers of Foxp3+ Treg. Our findings suggest a crucial role for Th17 cells in mediating airway B-cell influx and IgA response, and demonstrate that antigen-specific nTreg suppress Th17-mediated lung inflammation. These results provide new insights into how Th17 responses are limited and may facilitate development of novel approaches for controlling Th17-induced inflammation.

The role of SV40 in malignant mesothelioma and other human malignancies.

SV40 is a DNA tumor virus thrust upon human populations primarily as a contaminant in various vaccine preparations. Some estimates suggest that millions of people are currently infected with the virus. The virus causes primary brain tumors, bone tumors, lymphomas, and mesotheliomas when injected into some rodent models. It has also been detected in a similar spectrum of human tumors. However, epidemiological studies have failed to conclusively demonstrate a higher incidence of disease in affected populations. To date, over 60 reports from 49 different laboratories have shown SV40 sequences in tissues from human cancer patients. Six studies, however, have failed to detect evidence of virus in similar tissues. Some have suggested that SV40 may act as a cocarcinogen with asbestos to cause mesothelioma formation, or that it may be responsible for the 10-20% of mesotheliomas with no reported history of asbestos exposure. This report briefly covers the historical evidence for SV40 carcinogenesis and then covers experiments now underway to better understand the role of SV40 in human mesotheliomas.

Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates.

To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.