Epitope hierarchy of spontaneous CD4+ T cell responses to LAGE-1

NY-ESO-1 and LAGE-1 represent highly homologous cancer-germline Ags frequently coexpressed by many human cancers, but not by normal tissues, except testis. In contrast to NY-ESO-1, little is known about spontaneous immune responses to LAGE-1. In the current study, we report on spontaneous LAGE-1-specific CD4(+) T cells isolated from PBLs of patients with advanced LAGE-1(+)/NY-ESO-1(+) melanoma and directed against three promiscuous and immunodominant epitopes. Strikingly, although the three LAGE-1-derived epitopes are highly homologous to NY-ESO-1-derived epitopes, LAGE-1-specific CD4(+) T cells did not cross-react with NY-ESO-1. LAGE-1-specific CD4(+) T cells produced Th1-type and/or Th2-type cytokines and did not exert inhibitory effects on allogenic T cells. We observed that most patients with spontaneous NY-ESO-1-specific responses exhibited spontaneous CD4(+) T cell responses to at least one of the three immunodominant LAGE-1 epitopes. Additionally, nearly half of the patients with spontaneous LAGE-1-specific CD4(+) T cell responses had circulating LAGE-1-specific Abs that recognized epitopes located in the C-terminal portion of LAGE-1, which is distinct from NY-ESO-1. Collectively, our findings define the hierarchy of immunodominance of spontaneous LAGE-1-specific CD4(+) T cell responses in patients with advanced melanoma. These findings demonstrate the capability of LAGE-1 to stimulate integrated cellular and humoral immune responses that do not cross-react with NY-ESO-1. Therefore, they provide a strong rationale for the inclusion of LAGE-1 peptides or protein in vaccine trials for patients with NY-ESO-1(+)/LAGE-1(+) tumors.     

Transient finite element modeling of functional electrical stimulation

Transcutaneous functional electrical stimulation is commonly used for strengthening muscle. However, transient effects during stimulation are not yet well explored. The effect of an amplitude change of the stimulation can be described by static model, but there is no differency for different pulse duration. The aim of this study is to present the finite element (FE) model of a transient electrical stimulation on the forearm. Discrete FE equations were derived by using a standard Galerkin procedure. Different tissue conductive and dielectric properties are fitted using least square method and trial and error analysis from experimental measurement. This study showed that FE modeling of electrical stimulation can give the spatial-temporal distribution of applied current in the forearm. Three different cases were modeled with the same geometry but with different input of the current pulse, in order to fit the tissue properties by using transient FE analysis. All three cases were compared with experimental measurements of intramuscular voltage on one volunteer.     

Elucidating Novel Serum Biomarkers Associated with Pulmonary Tuberculosis Treatment

In an unbiased approach to biomarker discovery, we applied a highly multiplexed proteomic technology (SOMAscan, SomaLogic, Inc, Boulder, CO) to understand changes in proteins from paired serum samples at enrollment and after 8 weeks of TB treatment from 39 patients with pulmonary TB from Kampala, Uganda enrolled in the Center for Disease Control and Prevention’s Tuberculosis Trials Consortium (TBTC) Study 29. This work represents the first large-scale proteomic analysis employing modified DNA aptamers in a study of active tuberculosis (TB). We identified multiple proteins that exhibit significant expression differences during the intensive phase of TB therapy. There was enrichment for proteins in conserved networks of biological processes and function including antimicrobial defense, tissue healing and remodeling, acute phase response, pattern recognition, protease/anti-proteases, complement and coagulation cascade, apoptosis, immunity and inflammation pathways. Members of cytokine pathways such as interferon-gamma, while present, were not as highly represented as might have been predicted. The top proteins that changed between baseline and 8 weeks of therapy were TSP4, TIMP-2, SEPR, MRC-2, Antithrombin III, SAA, CRP, NPS-PLA2, LEAP-1, and LBP. The novel proteins elucidated in this work may provide new insights for understanding TB disease, its treatment and subsequent healing processes that occur in response to effective therapy.     

The involvement of proliferation and apoptosis in the early human gonad development

Distributions of the Ki-67, TP53, caspase-3 and AIFM1 markers were histologically investigated in the 5th to 9th week developing gonads of 12 human conceptuses using immunohistochemical and immunofluorescence methods. Between the 5th and 8th developmental week, proliferation gradually increased in the surface gonad epithelium (26–52 %) and stroma (19–42 %), but then slightly decreased in the surface epithelium (35 %) during the early foetal period. In medulla, low proliferation activity decreased from 15 to 12 % between the 7th and 9th week. At earliest stages of gonadal development, primordial germ cells (PGC) were only rarely TP53 positive. In the 7th and 8th week, almost all PGC-s displayed TP53 positivity, while their number decreased in early fetal period. During the investigated period, caspase-3 reactivity gradually decreased in surface epithelium, while it increased in PGC and medulla of developing gonad AIFM1-positivity first appeared in surface gonad epithelium and then predominantly in PCG-s while caspase-3 characterized different cell populations within the developing gonad. AIFM1 and caspase-3 co-localized only during the migration of PCG-s. The number and distribution of Ki-67, TP53, caspase-3 and AIFM1 reacting cells changed coincidently with development end regression of the sex cords in indifferent and early fetal gonad. Our results indicate that the number of PGC might be controlled by balance of TP53 and AIFM1, leading to caspase-3 independent cell death. Other cell populations are probably eliminated by caspase-3-dependent cell death. Both pathways of cell death seem to operate during early human gonad development, while their intensity varies depending on the cell type and developmental period analysed.     

The Sholl analysis of neuronal cell images: semi-log or log-log method?

Although the Sholl analysis is a quantitative method for morphometric neuronal studies and its application provides many benefits to neurobiology since it is obvious, common and meaningful, there are many unresolved theoretical issues that need to be addressed. Nevertheless, it can be practiced without much background or sophistication. The two different methods of the Sholl analysis--log-log and semi-log--have been applied previously without a clear basis as to what to use. To make an adequate choice of the method, one should try and accept that one which gives a better result. We consider that some of the underlying principles, assumptions and limitations for the Sholl analysis can be found in basic mathematics. In order to compare the results of applications of the semi-log and log-log methods to the same neuron, we introduce the concept of determination ratio as the ratio of the coefficient of determination for the semi-log method and that for the log-log method. If the semi-log method is better as related to the log-log method, the value of this parameter is larger than 1. Having in mind that dendrites exhibit enormously diverse forms, we point out that the semi-log Sholl method is more frequently utilizable in practice. Only the neurons, whose dendritic trees have one or a few sparsely ramified dendrites being much longer than the rest ones, could be successfully and exactly analysed using the log-log method. We also compare the Sholl analysis with fractal analysis for the characterization of neuronal arborization patterns and found that between the Sholl and fractal analysis exist various and important analogies.     

Biological control of Botrytis cinerea and plant growth promotion potential by Penicillium citrinum in chickpea (Cicer arietinum L.)

A total of 48 fungi were characterised for their antagonistic potential against Botrytis cinerea causing Botrytis Gray Mold (BGM) disease in chickpea by dual culture and metabolite production assays. The culture filtrate of the most promising isolate, VFI-51, was purified by various chromatographic techniques and identified as ‘citrinin’ by nuclear magnetic resonance and mass spectrometry studies. The efficacy of citrinin was demonstrated to control BGM in chickpea under greenhouse conditions. The sequences of 18S rDNA gene of the VFI-51 matched with Penicillium citrinum in BLAST analysis. The VFI-51 produced siderophore, hydrocyanic acid, indole-3-acetic acid, lipase, protease and β-1,3-glucanase; grew well in NaCl (up to 15%), at pH between 7 and 11 and temperatures between 20°C and 40°C; and was compatible with fungicides bavistin and thiram. Under greenhouse and field conditions, VFI-51 significantly enhanced the nodule number, nodule weight, root and shoot weight and stover and grain yield over the un-inoculated control. In the rhizosphere, VFI-51 also significantly enhanced total N, available P and OC over the un-inoculated control. Scanning electron microscopy analysis revealed that VFI-51 colonised on the roots of chickpea. This study concluded that VFI-51 has the potential for biocontrol of BGM and plant growth promotion in chickpea.     

Taxonomic notes on European taxa of Crataegus (Rosaceae)

In connection with systematic study of European taxa of Crataegus (Rosaceae subfam. Maloideae) lectotypes are designated for Crataegus monogyna f. subdigyna and C. monogyna var. ronnigeri. C. microphylla subsp. malýana is described as a taxon new to science and a key to the recognised taxa of C. microphylla and of the closely related C. rhipidophylla is given.     

The impact of football training on motor development in male children

The aim of the study was to determine the effect of football school program and physical education curriculum on changes in the motor abilities of 7- and 8-year-old boys. The study included a sample of 180 boys divided into group 1 (7-year-old boys), subdivided to experimental (n = 40) and control (n = 50) groups, and group 2 (8-year-old boys), subdivided to experimental (n = 40) and control (n = 50) groups. Experimental groups included children attending three training units of football training over a 9-month period, in addition to the conventional physical education curriculum. Control groups included children attending only conventional physical education curriculum. All study subjects underwent testing with a battery of 12 motor tests at the beginning and at the end of the study. Results obtained by discriminative canonic analysis showed no statistically significant between-group difference in motor abilities at the beginning of the study. However, significant differences in favor of experimental groups were recorded at the end of the study. Favorable changes in all motor variables were observed in both experimental and control groups of children from the initial through the final state. These changes were more pronounced in experimental groups. Analysis of variance for difference variables (final to initial measurement) indicated programmed education in the form of football training in addition to regular physical education curriculum to predominantly influence the development of aerobic endurance, agility, speed and flexibility in 7-year-old boys, and of explosive strength, aerobic endurance, flexibility and speed in 8-year-old boys. In the latter, football training led to the formation of a motor complex integrating explosiveness, speed, coordination, endurance and flexibility as a general motor factor determining future quality development in football.     

One NY-ESO-1-derived epitope that promiscuously binds to multiple HLA-DR and HLA-DP4 molecules and stimulates autologous CD4+ T cells from patients with NY-ESO-1-expressing melanoma

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87-111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87-111 stimulates Th1-type and Th-2/Th0-type CD4(+) T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4(+) T cells and CD4(+) T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-gamma and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4(+) T cells recognizing peptide NY-ESO-1 87-111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87-111 in the large number of cancer patients with NY-ESO-1-expressing tumors.     

DNA polymerase III holoenzyme from Thermus thermophilus identification, expression, purification of components, and use to reconstitute a processive replicase

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the beta sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of alpha (pol III catalytic subunit), beta (sliding clamp processivity factor), and the essential DnaX (tau/gamma), delta and delta' components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including delta-delta' interaction, deltadelta'-tau/gamma complex formation, and alpha-tau interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 degrees C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 degrees C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.