Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-RI) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis, In previous studies, me have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis, We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Pour of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors, (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-RI and more so for the TRAIL decoy receptors TRAIL-R3 and -R4, Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-RI and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-RI and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alpha A-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alpha ACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alpha ACE6E7#19 mice, but not to nontransgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised aACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alpha ACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alpha ACE6E7#19 mice was observed in scid-/- aACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines a re therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Human and murine cytomegalovirus evasion of cytotoxic T lymphocyte and natural killer cell-mediated immune responses

Herpesviruses, such as human and murine cytomegalovirus, possess an impressive array of genes believed to assist in virus survival against the host immune response. In this review, we cover the rapidly growing area of cytomegalovirus evasion of cellular immunity, specifically cytotoxic T lymphocytes and natural killer cells. The proposed mechanisms of action of viral proteins involved in blocking peptide presentation to CD8(+) T cells, namely, interference with peptide generation, inhibition of peptide assembly with class I MHC and retention/destabilization of class I MHC complexes, are described. In addition, recent evidence implicating the viral class I MHC-like proteins as inhibitors of natural killer cell-mediated clearance is reviewed, (C) 1998 Academic Press.

Engineered detoxification confers resistance against a pathogenic bacterium

We generated transgenic sugarcane plants that express an albicidin detoxifying gene (albD), which was cloned from a bacterium that provides biocontrol against leaf scald disease. Plants with albicidin detoxification capacity equivalent to 1-10 ng of AlbD enzyme per mg of leaf protein did not develop chlorotic disease symptoms in inoculated leaves, whereas all untransformed control plants developed severe symptoms. Transgenic lines with high AlbD activity in young stems were also protected against systemic multiplication of the pathogen, which is the precursor to economic disease. We have shown that genetic modification to express a toxin-resistance gene can confer resistance to both disease symptoms and multiplication of a toxigenic pathogen in its host.

Monooxygenases play only a minor role in resistance to synthetic pyrethroids in the cattle tick, Boophilus microplus

We investigated the role of monooxygenases in resistance to synthetic pyrethroids (SPs) in the cattle tick, Boophilus microplus. We found that monooxygenases play only a minor role in resistance to SPs in both resistant and susceptible strains of B. microplus. We blocked the monooxygenases with piperonyl butoxide (PBO) and simultaneously applied the SPs, flumethrin and cypermethrin to larval B. microplus. PBO increased the effect of flumethrin (synergism ratios 2.7-8.9) more than it increased the effect of cypermethrin (synergism ratios 1.9-3.1). Of the four strains tested, Parkhurst, which is resistant to SPs, was the least affected by the addition of PBO (synergism ratios after cypermethrin was applied 1.9; after flumethrin 2.7) whereas N.R.F.S., the strain susceptible to SPs, was the most affected by synergism between PBO and SPs (synergism ratio after cypermethrin was applied 3.1; after flumethrin 8.9). We hypothesize that B. microplus lacks monooxygenases capable of conferring resistance to SPs because it and its recent ancestors were blood-feeders rather than herbivores.

Acute susceptibility of aged mice to infection with Candida albicans

The effect of aging on host resistance to systemic candidosis was assessed by monitoring the course of infection in 16-month-old CBA/CaH mice (aged non-immune) and in a comparable group that had been infected with a sublethal dose of Candida albicans at 6 weeks of age (aged immune). Aged non-immune mice showed rapid progression of the disease, with a marked increase in the number of mycelia in the brain and kidney, and early morbidity, Foci of myocardial necrosis were evident, but inflammatory cells were sparse. The histological picture in the aged immune mice was similar to that in the aged non-immune group, although fewer mycelial aggregates were seen. Both groups of aged mice showed a significantly lower fungal burden in the brain on day 1 of infection, but on day 4, colony counts increased significantly in the aged non-immune mice, Comparison of cytokine gene expression in the infected brains showed that the relative amount of interferon-gamma and tumour necrosis factor-alpha cDNA were similar in all three groups. Interleukin-6 was elevated in both infected non-immune and uninfected aged mice. Aged immune mice showed no morbidity after challenge, and both colonisation and tissue damage were reduced in comparison with the aged non-immune animals.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Perforin-mediated cytotoxicity is critical for surveillance of spontaneous lymphoma

Immune surveillance by cytotoxic lymphocytes against cancer has been postulated for decades, but direct evidence for the role of cytotoxic lymphocytes in protecting against spontaneous malignancy has been lacking. As the rejection of many experimental cancers by cytotoxic T lymphocytes and natural killer cells is dependent on the pore-forming protein perforin (pfp), we examined pfp-deficient mice for increased cancer susceptibility. Here we show that pfp-deficient mice have a high incidence of malignancy in distinct lymphoid cell lineages (T, B, NKT), indicating a specific requirement for pfp in protection against lymphomagenesis. The susceptibility to lymphoma was accentuated by simultaneous lack of expression of the p53 gene, mutations in which also commonly predispose to human malignancies, including lymphoma. In contrast, the incidence and age of onset of sarcoma was unaffected in p53-deficient mice. Pfp-deficient mice were at least 1,000-fold more susceptible to these lymphomas when transplanted, compared with immunocompetent mice in which tumor rejection was controlled by CD8(+) T lymphocytes. This study is the first that implicates direct cytotoxicity by lymphocytes in regulating lymphomagenesis.

Glutamate-mediated transmission, alcohol, and alcoholism

Glutamate-mediated neurotransmission may be involved in the range of adaptive changes in brain which occur after ethanol administration in laboratory animals, and in chronic alcoholism in human cases. Excitatory amino acid transmission is modulated by a complex system of receptors and other effecters, the efficacy of which can be profoundly affected by altered gene or protein expression. Local variations in receptor composition may underlie intrinsic regional variations in susceptibility to pathological change. Equally, ethanol use and abuse may bring about alterations in receptor subunit expression as the essence of the adaptive response. Such considerations may underlie the regional localization characteristic of the pathogenesis of alcoholic brain damage, or they may form part of the homeostatic change that constitutes the neural substrate for alcohol dependence. (C) 2000 Elsevier Science Ltd. All rights reserved.

Resistance to temperature extremes between and within life cycle stages in Drosophila serrata, D-birchii and their hybrids: intraspecific and interspecific comparisons

Intraspecific Drosophila studies suggest that resistance to heal and cold stresses are largely independent and that correlations across life cycle stages are low whereas comparisons of Drosophila species indicate correlations between heat and cold resistance as well as between resistance levels in different life cycle stages. These inconsistent results may reflect differences in associations among traits at the interspecific and intraspecific levels or interspecific correlations arising because of correlated selection pressures. These alternatives were tested using Drosophila serrata, D. birchii and hybrids derived from these species. Variation among hybrid lines and families was used to test associations at the interspecific level while intraspecific variation was examined using isofemale lines of D. serrata. There was a significant association between adult heat knockdown time at 38 degreesC and adult cold resistance in one set of hybrid lines. An association between female knockdown resistance to heat and larval heat resistance was also evident in one set of hybrids. Resistance to heat anti cold at the larval stage were not correlated at either the intraspecific or interspecific levels. At the intraspecific level, lan al heat resistance and two measures of adult heat resistance were uncorrelated. Moreover, adult and larval cold resistance measures were not correlated at either the intraspecific or interspecific levels. These results suggest that there are no associations between resistance to heat and cold extremes and that extreme temperature resistance is largely independent across life cycle stages at both the intraspecific and interspecific levels. Species associations may therefore arise from correlated selection pressures rather than trait correlations. (C) 2000 The Linnean Society of London.

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for CA-binding protein (GABP)

Within steroid receptor heterocomplexes the large tetraticopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (HspSO) and act coordinately with HspSO to modulate receptor activity. The reversible nature of the interaction between the immunophilins and HspSO suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a fi-kilobase (kb) 5 ' -flanking region of the human gene and demonstrated that a similar to 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GAFF is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Lentiviral vector-mediated tyro sinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), which play a vital role in primary immune responses. Introducing genes into DCs will allow constitutive expression of the encoded proteins and thus prolong the presentation of the antigens derived therefrom. In addition, multiple and unidentified epitopes encoded by the entire tumor-associated antigen (TAA) gene may enhance T cell activation. This study demonstrated that an HIV-1-based lentiviral vector conferred efficient gene transfer to DCs. The transgene, murine tyrosinase-related protein 2 (mTRP-2), encodes a clinically relevant melanoma-associated antigen (MAA), which has been found to be a tumor rejection antigen for B16 melanoma. The transfer and proper processing of mTRP-2 in DCs, in terms of RNA transcription activity and protein expression, were verified by RT-PCR and specific antibody, respectively. Administration of mTRP-2 gene-modified DCs (DC-HR'CmT2) to C57BL/6 mice evoked strong protection against tumor challenge, for which the presence of CD4(+) and CD8(+) cells during both the priming and challenge phase was essential. In a therapy model, our results showed that four of seven mice with preestablished tumor remained tumor free for 80 days after therapeutic vaccination. Given the results shown in this study, mTRP-2 gene transfer to DCs provides a potential therapeutic strategy for the management of melanoma, especially in the early stage of the disease.

Resistance to UVB radiation in Antarctic yeasts

Yeast cells were used as a model system to study the inter-relationship among free radicals, antioxidants and UV-induced cell damage. In particular, the effects of UV-radiation in newly isolated yeasts from the Antarctic have been studied.