Polyclonal T-cell activation protocol stimulates preferential expansion of EBV-specific T-cell clones in vitro

Purpose Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukemia. The most important anti-leukemic effect may be mediated by the T-cells contained within the graft; however, the allogeneic T-cells may also give rise to graft-vs-host disease (GVHD). One way to control GVHD might be to transduce the donor T-cells with a drug-inducible suicide gene. If a retrovirus vector is to be used for this transduction, activation of the T-cells is required for integration of the transgene to occur. The activation protocol should ensure expansion of a broad repertoire of donor T-cells. Notably, T-cells specific for herpes virus family antigens are important for adoptive immunoprotection.Methods To define optimal activation conditions for retrovirus-mediated suicide gene transduction of donor T-cells, we examined the repertoire of CD8+ T-cells in general, and Epstein-Barr virus (EBV) specific T-cells in particular, following two different activation and expansion procedures.Results We found that repeated CD3/CD28 stimulation resulted in a high level of activation-induced T-cell death, affecting in vivo expanded clones, some of which were specific for EBV, in particular. In contrast, initial CD3/CD28 activation followed by proliferation in interleukin-2 lead to expansion of EBV-specific clones over and above the expansion observed for CD8+ T-cells in general.Conclusion These results should impact on protocols for ex vivo activation of T-cells prior to suicide gene transduction.     

Functional HLA-DR T cell epitopes of CEA identified in patients with colorectal carcinoma immunized with the recombinant protein CEA

A baculovirus-produced recombinant CEA (rCEA) protein comprising the extracellular region was used for vaccination of CRC patients with or without GM-CSF as an adjuvant cytokine. Ten patients with a significant proliferative T cell response against rCEA were selected for T cell epitope mapping. Fifteen-aa-long overlapping peptides covering the entire aa sequence of the external domain of CEA were used in a proliferation assay. In six of the patients a repeatable T cell response against at least one peptide was demonstrated. For the first time, nine functional HLA-DR epitopes of CEA were defined. Two of the peptides were recognized by more than one patient, i.e., two and three patients, respectively. Those 15-mer peptides that induced a proliferative T cell response fitted to the actual HLA-DR type (SYFPEITHI). The affinity of the native peptides for the T cell receptor was in the low to intermediate range (scores 6–19). The 15-mer peptides also contained 9-mer peptide sequences that could be predicted to bind to the actual HLA-ABC genotypes (SYFPEITHI/BIMAS). Blocking experiments using monoclonal antibodies indicated that the proliferative T cell response was both MHC class I and II restricted. The defined HLA-DR T cell epitopes were spread over the entire CEA molecule, but a higher frequency was noted towards the C-terminal. Peptides with a dual specificity may form a basis for production of subunit cancer vaccines, but modifications should be done to increase the T cell affinity, thereby optimizing the antitumoral effects of the vaccine.Abbreviations aa amino acid - CRC colorectal carcinoma - GM-CSF granulocyte/monocyte colony stimulating factor - CEA carcino-embryonic antigen - BCP baculovirus control protein - MHC major histocompatibility complex - pp peptide - TAA tumor associated antigen     

Exploring structural features of the interaction between the scorpion toxinCnErg1 and ERG K+ channels

The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruro?des noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.     

Microtubules become more dynamic but not shorter during preprophase band formation: a possible "search-and-capture" mechanism for microtubule translocation

The dynamic behavior of the microtubule cytoskeleton plays a crucial role in cellular organization, but the physical mechanisms underlying microtubule (re)organization in plant cells are poorly understood. We investigated microtubule dynamics in tobacco BY-2 suspension cells during interphase and during the formation of the preprophase band (PPB), the cytoskeletal structure that defines the site of cytokinesis. Here we show that after 2 h of microtubule accumulation in the PPB and concurrent disappearance elsewhere in the cortex, the PPB is completed and starts to breakdown exponentially already 20 min before the onset of prometaphase. During formation of the PPB, the dynamic instability, i.e., the stochastic alternating between growing and shrinking phases, of the cortical microtubules outside the PPB increases significantly, but the microtubules do not become shorter. Based on this, as well as on the cross-linking of microtubules in the PPB and the lack of evidence for motor involvement, we propose a "search-and-capture" mechanism for PPB formation, in which the regulation of dynamic instability causes the cortical microtubules to become more dynamic and possibly longer, while the microtubule cross-linking activity of the developing PPB preferentially stabilizes these "searching" microtubules. Thus, microtubules gradually disappear from the cortex outside the PPB and aggregate to the forming PPB.     

Increased cytochrome P450 17alpha-hydroxylase promoter function in theca cells isolated from patients with polycystic ovary syndrome involves nuclear factor-1

Cytochrome P450 17alpha-hydroxylase (CYP17) gene expression and androgen biosynthesis are persistently elevated in theca cells isolated from ovaries of women with polycystic ovary syndrome (PCOS). We previously reported that -235 to -109 bp of the CYP17 promoter confers increased CYP17 promoter function in PCOS theca cells. In this report, additional deletion and mutational analyses of the CYP17 promoter were performed to identify the sequences that contribute to increased CYP17 promoter function in PCOS theca cells. Results of these analyses established that augmented promoter function in PCOS theca cells results from preferentially increased basal regulation conferred by sequences between -188 and -147 bp of the CYP17 promoter. Scanning mutant analysis demonstrated that mutations within a 16-bp sequence, spanning -174 to -158 bp of the promoter, ablated increased basal CYP17 promoter function in PCOS theca cells. EMSA analysis demonstrated that the NF-1 family member, NF-1C, bound this sequence. Cotransfection of several NF-1C isoforms expressed in normal and PCOS cells repressed CYP17 promoter function. NF-1C protein and DNA binding were reduced in PCOS theca cell nuclear extracts, as compared with normal. Another NF-1C site between -102 and -90 bp of the promoter was also identified. However, mutation of this site had no effect on differential promoter function in PCOS theca cells. These studies demonstrate that 1) augmented CYP17 promoter function in PCOS theca cells results from increased basal regulation, and 2) diminished NF-1C-dependent repression may be one mechanism underlying increased basal CYP17 promoter activity and altered gene expression in PCOS theca cells.     

Depressed thermogenesis but competent brown adipose tissue recruitment in mice devoid of all hormone-binding thyroid hormone receptors

We have examined the metabolic role of hormone-binding nuclear thyroid hormone receptors (TRs). Mice devoid of all hormone-binding TRs [TR alpha 1(-/-)beta(-/-) (TR-ablated mice)] had slightly decreased body temperature and much decreased basal metabolic rate, were still able to markedly increase metabolic rate in the cold, but were cold intolerant due to inadequate total heat production at low temperatures. A standard norepinephrine test showed that adrenergically induced thermogenesis could not be activated normally in the TR-ablated mice. This was not due to inadequate recruitment of brown adipose tissue, nor to the absence, decreased recruitment or dysfunction of the uncoupling protein-1. However, isolated brown fat cells were 10-fold desensitized, explaining the lack of response to standard adrenergic stimuli; cell culture experiments demonstrated that this desensitization was not an innate effect. Thus, the cold intolerance was probably not due to inadequate sympathetically induced nonshivering thermogenesis. Additionally, the results indicated that no metabolic effects of thyroid hormones could become manifest in the absence of nuclear TRs, that ligand-bound TRs were needed for euthermia and eumetabolism, but that TRs per se were not required for brown adipose tissue recruitment and uncoupling protein-1 gene expression.     

Solar lentigines are strongly related to sun exposure in contrast to ephelides

Solar lentigines and ephelides are different types of pigmented skin lesions predominantly present on sun-exposed skin. Both lesions are risk indicators for melanoma and non-melanoma skin cancer. Solar lentigines are considered as a sign of photodamage although well-conducted epidemiological studies are lacking on this subject. Ephelides are associated with fair skin type and red hair. The aim of the present study was to investigate the relation of sun-exposure estimates with solar lentigines and ephelides. In the Leiden Skin cancer Study 577 patients with malignant melanoma and/or non-melanoma skin cancer and 385 individuals without a history of skin cancer were studied. The presence of solar lentigines and ephelides in the face and on the back was assessed. Data on skin type, hair color, sun-exposure variables and cutaneous signs of photodamage were collected, by questionnaire and physical examination. Data were analyzed by chi-square or Student t-tests and with multivariable regression. Exposure odds ratios with 95% confidence intervals (95% CI) were calculated to estimate the relative risk for the presence of solar lentigines and ephelides dependent on signs of photodamage. The association with age was strongly positive for solar lentigines whereas it was strongly negative for ephelides (P-values for trend <0.0001). After adjustment for age, sex and skin type, solar lentigines on the back were positively associated with cumulative (P = 0.01) and intermittent (P = 0.0002) sun exposure. After adjustment, solar lentigines on the back were also associated with a history of sunburns before the age of 20 yr (P = 0.0003) and the number of sunburns in childhood (P = 0.002). Solar lentigines in the face were significantly associated with cutaneous signs of photodamage, i.e. elastosis (odds ratio 2.4, 95% CI 1.7-3.3) and actinic keratosis (odds ratio 1.8, 95% CI 1.3-2.4) whereas ephelides were not. Ephelides in the face and on the back showed an inverse association with chronic sun exposure but after adjustment theses associations disappeared. Sunburns before the age of 20 appeared to be positively associated with ephelides on the back (P = 0.04). In contrast to lentigines, ephelides were much more associated with constitutional host factors such as fair skin and/or red hair (both P < 0.0001). This study indicates that both chronic and acute sun exposure are important in the pathogenesis of solar lentigines.     

Letter to the Editor: 1H, 15N and 13C NMR Resonance Assignments of Staphostatin A, a Specific Staphylococcus Aureus Cysteine Proteinase Inhibitor

The increasing antibiotic resistance of an important human pathogen Staphylococcus aureus calls for the development of new therapeutic strategies. Staphylococcal cysteine proteases have been suggested as targets for such therapies. The recent discovery of staphostatins, specific protein inhibitors of these enzymes, gives prospects for the design and production of synthetic, low molecular weight analogs which might become drugs. We have decided to structurally characterize staphostatin A, a representative inhibitor of staphylococcal cysteine proteases, and to assess its binding mode to the target protease with the view of clarifying the specificity determinants. Here we report the (1)H, (15)N and (13)C NMR resonance assignments of staphostatin A.