Combining near-infrared-excited autofluorescence and Raman spectroscopy improves in vivo diagnosis of gastric cancer

This study aims to evaluate the diagnostic utility of the combined near-infrared (NIR) autofluorescence (AF) and Raman spectroscopy for improving in vivo detection of gastric cancer at clinical gastroscopy. A rapid Raman endoscopic technique was employed for in vivo spectroscopic measurements of normal (n=1098) and cancer (n=140) gastric tissues from 81 gastric patients. The composite NIR AF and Raman spectra in the range of 800-1800 cm(-1) were analyzed using principal component analysis (PCA) and linear discriminant (LDA) to extract diagnostic information associated with distinctive spectroscopic processes of gastric malignancies. High quality in vivo composite NIR AF and Raman spectra can routinely be acquired from the gastric within 0.5s. The integrated intensity over the range of 800-1800 cm(-1) established the diagnostic implications (p=1.6E-14) of the change of NIR AF intensity associated with neoplastic transformation. PCA-LDA diagnostic modeling on the in vivo tissue NIR AF and Raman spectra acquired yielded a diagnostic accuracy of 92.2% (sensitivity of 97.9% and specificity of 91.5%) for identifying gastric cancer from normal tissue. The integration area under the receiver operating characteristic (ROC) curve using the combined NIR AF and Raman spectroscopy was 0.985, which is superior to either the Raman spectroscopy or NIR AF spectroscopy alone. This work demonstrates that the complementary Raman and NIR AF spectroscopy techniques can be integrated together for improving the in vivo diagnosis and detection of gastric cancer at endoscopy.     

Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer

Background aimsAdoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies.MethodsIn a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr⁵¹ release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion.ResultsTIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8⁺ and CD4⁺ subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells.ConclusionThe procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.     

Harbour porpoises respond to climate change

The effects of climate change on marine ecosystems and in particular on marine top predators are difficult to assess due to, among other things, spatial variability, and lack of clear delineation of marine habitats. The banks of West Greenland are located in a climate sensitive area and are likely to elicit pronounced responses to oceanographic changes in the North Atlantic. The recent increase in sea temperatures on the banks of West Greenland has had cascading effects on sea ice coverage, residency of top predators, and abundance of important prey species like Atlantic cod (Gadus morhua). Here, we report on the response of one of the top predators in West Greenland; the harbour porpoise (Phocoena phocoena). The porpoises depend on locating high densities of prey species with high nutritive value and they have apparently responded to the general warming on the banks of West Greenland by longer residence times, increased consumption of Atlantic cod resulting in improved body condition in the form of larger fat deposits in blubber, compared to the situation during a cold period in the 1990s. This is one of the few examples of a measurable effect of climate change on a marine mammal population.     

Spontaneous T-cell responses against peptides derived from the Taxol resistance–associated gene-3 (TRAG-3) protein in cancer patients

Expression of the cancer-testis antigen Taxol resistance–associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01–restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3–derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01–binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3–specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2⁺ donors, but no responses were detectable in 9 HLA-A2^– healthy donors or 4 HLA-A2^– melanoma patients. The identified HLA-A*02.01–restricted TRAG-3–derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.     

Characterization of an allosteric citalopram-binding site at the serotonin transporter

The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. In the present study we show that the dissociation rate, of [3H]S-citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/- 0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [3H]S-citalopram. Allosteric modulation of dissociation is independent of temperature, or the presence of Na+ in the dissociation buffer. Dissociation of [3H]S-citalopram from a complex with the SERT double-mutant, N208Q/N217Q, which has been suggested to be unable to self-assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild-type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species-scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high-affinity binding at the primary site.     

Seroprevalence and risk factors for Helicobacter pylori infection in Greenlanders

BACKGROUND: In contrast to most populations worldwide, the incidence of gastric cancer increases among Inuit in Greenland. Contributing factors to this increase are unknown, but Helicobacter pylori may be involved. However, little is known regarding the epidemiology of H. pylori in Arctic communities. With the aim of determining age-specific prevalence, risk factors, and association with clinical conditions of H. pylori infection, we carried out a population-based study of H. pylori in Sisimiut, the second biggest town of Greenland. MATERIALS AND METHODS: A population-based sample of 685 persons had serum drawn that was analyzed for H. pylori IgG antibodies using enzyme-linked immunosorbent assay (ELISA). Risk factors analyses were carried out using multivariate logistic regression models. RESULTS: The seroprevalence was lowest among children aged 0-4 years (6%), but increased rapidly thereafter. In persons aged 15-87 years the seroprevalence had stabilized around 58%. Total number of children in household, number of older, but not younger, siblings and narrow age gap to closest older sibling were associated with H. pylori seropositivity. In contrast, number of adults in household and socioeconomic status did not influence serostatus. CONCLUSIONS: The age-specific prevalence pattern in Greenland is intermediate between that of developing and developed countries. The risk factor pattern indicates crowding and older siblings in particular to be key elements in risk of infection.     

On the mechanisms of glycolytic oscillations in yeast

This work concerns the cause of glycolytic oscillations in yeast. We analyse experimental data as well as models in two distinct cases: the relaxation-like oscillations seen in yeast extracts, and the sinusoidal Hopf oscillations seen in intact yeast cells. In the case of yeast extracts, we use flux-change plots and model analyses to establish that the oscillations are driven by on/off switching of phosphofructokinase. In the case of intact yeast cells, we find that the instability leading to the appearance of oscillations is caused by the stoichiometry of the ATP-ADP-AMP system and the allosteric regulation of phosphofructokinase, whereas frequency control is distributed over the reaction network. Notably, the NAD+/NADH ratio modulates the frequency of the oscillations without affecting the instability. This is important for understanding the mutual synchronization of oscillations in the individual yeast cells, as synchronization is believed to occur via acetaldehyde, which in turn affects the frequency of oscillations by changing this ratio.     

Interaction between the catalytic site and the A-M3 linker stabilizes E2/E2P conformational states of Na+,K+-ATPase

The consequences of mutations Ile(265) --> Ala, Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala for the partial reaction steps of the Na(+),K(+)-ATPase transport cycle were analyzed. The mutated residues are part of the long loop ("A-M3 linker") connecting the cytoplasmic A-domain with transmembrane segment M3. It was found that mutation Ile(265) --> Ala displaces the E(1)-E(2) and E(1)P-E(2)P equilibria in favor of E(1)/E(1)P, whereas mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala displace these conformational equilibria in favor of E(2)/E(2)P. The mutations affect both the rearrangement of the cytoplasmic domains (seen by changes in phosphoenzyme properties and apparent ATP/vanadate affinities) and the membrane sector (indicated by change in K(+)/Rb(+) deocclusion rate). Destabilization of E(2)/E(2)P in Ile(265) --> Ala, as well as a direct effect on the intrinsic affinity of the E(2) form for vanadate, may be explained on the basis of the E(2) crystal structures of the Ca(2+)-ATPase, showing interaction of the equivalent isoleucine with conserved residues near the catalytic region of the P-domain. The rate of phosphorylation from ATP was unaffected in Ile(265) --> Ala, indicating a lack of interference with the catalytic function in E(1)/E(1)P. The effects of mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala provide the first evidence in the literature of a relative stabilization of E(2)/E(2)P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E(1)/E(1)P, increased stability of the A3 helix of the A-M3 linker in E(2)/E(2)P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain.