CD8 infiltration is associated with disease control and tobacco exposure in intermediate-risk oropharyngeal cancer.

Oropharyngeal squamous cell carcinoma (OPSCC) incidence is increasing at a nearly epidemic rate, largely driven by the human papillomavirus (HPV). Despite the generally favorable clinical outcomes of patients with HPV driven (HPV+) OPSCC, a significant subset of HPV tumors associated with tobacco exposure have diminished treatment response and worse survival. The tumor immune microenvironment (TIME) has been shown to be a critical driver of treatment response and oncologic outcomes in OPSCC generally and HPV+ OPSCC more specifically. However, the impact of tobacco exposure on the TIME in OPSCC patients remains unclear. We analyzed the relationship between TIME, tobacco exposure and clinical outcomes in OPSCC patients (n = 143) with extensive tobacco exposure (median pack-years = 40). P16 overexpression, a surrogate marker of HPV association, was a strong predictor of relapse-free (RFS) and overall survival (OS) (p < 0.001, p < 0.001 respectively) regardless of tobacco exposure and associated strongly with differential infiltration of the tumor by both CD3 and CD8 lymphocytes measured via immunohistochemistry (p < 001, p < 0.001 respectively). CD3 and CD8 infiltration was a strong predictor of RFS and OS and associated strongly with disease stage (AJCC 8th Edition Staging Manual). Tobacco exposure correlated significantly (p < 0.001) with decreased CD8 infiltration in p16+ OPSCC tumors. Our findings demonstrate that the HPV+ OPSCC clinical outcomes are strongly correlated with the TIME, which is potentially modulated by tobacco exposure. Immunomodulatory strategies targeting this disease in smokers must take into consideration the potential modifying effects of tobacco exposure on treatment effectiveness and clinical outcomes.

Truncated Gaussian-Bessel beams for short-pulse processing of small-aspect-ratio micro-channels in dielectrics.

In order to control the length of micro-channels ablated at the surface of dielectrics, we use annular filtering apertures for tailoring the depth of focus of micrometric Gaussian-Bessel beams. We identify experimentally and numerically the appropriate beam truncation that promotes a smooth axial distribution of intensity with a small elongation, suitable for processing micro-channels of small aspect ratio. Single-shot channel fabrication is demonstrated on the front surface of a fused silica sample, with sub-micron diameter, high-quality opening, and depth of few micrometers, using 1 ps low-energy (< 0.45 µJ) pulse. Finally, we realize 10 × 10 matrices of densely packed channels with aspect ratio ~5 and a spatial period down to 1.5 µm, as a prospective demonstration of direct laser fabrication of 2D photonic-crystal structures.

Experimental vaccine induces Th1-driven immune responses and resistance to Neisseria gonorrhoeae infection in a murine model.

Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microencapsulated interleukin-12 (IL-12), and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10-13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6-9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal immunoglobulin G (IgG) and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4+ T cells secreted interferon-γ (IFNγ), but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as elongation factor-Tu (EF-Tu) and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a nonliving gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate type 1 T helper cell (Th1)-driven immune responses in the genital tract.

Isomorphic coalescence of aster cores formed in vitro from microtubules and kinesin motors.

We report fluorescence microscopy studies of the formation of aster-like structures emerging from a cellular element-based active system and a novel analysis of the aster condensation. The system consists of rhodamine labeled microtubules which are dynamically coupled by functionalized kinesin motor proteins cross-linked via streptavidin-coated quantum dots (QDs). The aster-shaped objects contain core structures. The cores are aggregates of the QD-motor protein complexes, and result from the dynamic condensation of sub-clusters that are connected to each other randomly. The structural specificity of the aster core reflects a configuration of the initial connectivity between sub-clusters. Detailed image analysis allows us to extract a novel correlation between the condensation speed and the sub-cluster separation. The size of the core is scaled down during the condensation process, following a power law dependence on the distance between sub-clusters. The exponent of the power law is close to two, as expected from a geometric model. This single exponent common to all the contractile lines implies that there exists a time regime during which an isomorphic contraction of the aster core continues during the condensation process. We analyze the observed contraction by using a model system with potential applicability in a wide range of emergent phenomena in randomly coupled active networks, which are prevalent in the cellular environment.

Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies.

Within a decade the family of AlkB dioxygenases has been extensively studied as a one-protein DNA/RNA repair system in Escherichia coli but also as a group of proteins of much wider functions in eukaryotes. Two strains, HK82 and BS87, are the most commonly used E. coli strains for the alkB gene mutations. The aim of this study was to assess the usefulness of these alkB mutants in different aspects of research on AlkB dioxygenases that function not only in alkylated DNA repair but also in other metabolic processes in cells. Using of HK82 and BS87 strains, we found the following differences among these alkB(-) derivatives: (i) HK82 has shown more than 10-fold higher MMS-induced mutagenesis in comparison to BS87; (ii) different specificity of Arg(+) revertants; (iii) increased induction of SOS and Ada responses in HK82; (iv) the genome of HK82, in comparison to AB1157 and BS87, contains additional mutations: nalA, sbcC, and nuoC. We hypothesize that in HK82 these mutations, together with the non-functional AlkB protein, may result in much higher contents of ssDNA, thus higher in comparison to BS87 MMS-induced mutagenesis. In the light of our findings, we strongly recommend using BS87 strain in AlkB research as HK82, bearing several additional mutations in its genome, is not an exact derivative of the AB1157 strain, and shows additional features that may disturb proper interpretation of obtained results.

Popular species of edible mushrooms as a good source of zinc to be released to artificial digestive juices.

Because fruiting bodies of edible mushrooms accumulate elements very effectively, in this study for the first time we aimed at determining the degree of the release of zinc(II) ions to artificial digestive juices imitating the human gastrointestinal tract from freeze-dried popular edible mushroom fruiting bodies, such as Agaricus bisporus, Boletus badius and Cantharellus cibarius. For the analysis, anodic stripping voltammetry method was used. The amount of zinc released to artificial saliva within 1 minute ranged from 0.03 to 1.14 mg/100 g d.w. In gastric juice, the amounts were higher and ranged from 0.75 to 2.07 mg/100 g d.w. depending on the incubation time. After incubation of the freeze-dried edible mushroom fruiting bodies for 1 minute in artificial saliva, 15 in artificial gastric juice and then 150 minutes in artificial intestinal juice, it was found that the concentration of the released zinc in artificial intestinal juice was the highest and amounted to 6.44 mg/100 g d.w. The total average amount of zinc released from Boletus badius was the highest and this was estimated at 4.13 mg/100 g d.w. For the remaining two investigated species of A. bisporus and C. cibarius, the total amounts of zinc released into artificial digestive juices were only slightly lower and were estimated at 2.23 and 3.29 mg/100 g d.w. on average, respectively. It was demonstrated for the first time that mushrooms release zinc to artificial digestive juices imitating conditions in the human digestive tract and are a good source of this element.

Phenotype and function of nasal dendritic cells.

Intranasal (i.n.) vaccination generates immunity across local, regional, and distant sites. However, nasal dendritic cells (DCs), pivotal for the induction of i.n. vaccine-induced immune responses, have not been studied in detail. Here, by using a variety of parameters, we define nasal DCs in mice and humans. Distinct subsets of "classical" DCs, dependent on the transcription factor zbtb46 were identified in the murine nose. The murine nasal DCs were Fms-related tyrosine 3 kinase ligand responsive and displayed unique phenotypic and functional characteristics, including the ability to present antigen, induce an allogeneic T-cell response, and migrate in response to lipopolysaccharide or live bacterial pathogens. Importantly, in a cohort of human volunteers, BDCA-1(+) DCs were observed to be the dominant nasal DC population at steady state. During chronic inflammation, the frequency of both BDCA-1(+) and BDCA-3(hi) DCs was reduced in the nasal tissue, associating the loss of these immune sentinels with chronic nasal inflammation. The present study is the first detailed description of the phenotypic, ontogenetic, and functional properties of nasal DCs, and will inform the design of preventative immunization strategies as well as therapeutic modalities against chronic rhinosinusitis.

Activity of hospital transplant coordinators in Malopolskie Province (Poland).

BACKGROUND: In Poland as well as in other European countries, the number of organs from deceased donors is too small to meet the needs of transplantation therapy. METHODS: This situation can be improved by increasing the number of hospital transplant coordinators in hospitals with potential of donation. Since 2010, 200 Polish hospitals have employed coordinators whose role is to recruit deceased organ donors, to monitor the potential of donation (quality assurance program), and to run the training courses. In Malopolskie Province, there are 26 hospitals in which organ procurement from brain-dead donors is possible. In 13 hospitals, donor transplant coordinators have been employed. The objective of this study was to evaluate the activity of hospitals in Malopolskie Province in the field of donor recruitment before and after employment of coordinators (19 months before and after). For the purpose of the study, the number of hospitals with positive effects and with no effects of coordinator employment was calculated, and several donation rates were compared in the period before and after employment of the coordinator. We also compared the number of deceased organ donors in 13 hospitals employing a coordinator and in 13 hospitals without a coordinator. RESULTS: The desired impact of employment of coordinators in Malopolskie Province measured by improvement of organ donation rates was observed in half of the hospitals (7 of 13; 54%) with a transplant donor coordinator. The number of potential organ donors increased by 100% (from 24 to 48), and the number of actual organ donors increased by 113% (from 16 to 34). The percentage of family objections to organ donation decreased (from 17% to 8%). The best result of employing coordinators was observed at university hospitals and multidisciplinary hospitals and at hospitals in which the coordinator was a physician. The worst effect was recorded at county hospitals. CONCLUSIONS: The employment of hospital transplant coordinators in Malopolskie Province has a global impact on the increase of the number of actual organ donors in that region and improvement of organ donation rates, but it is effective only in half of the hospitals with coordinators. It indicates that other measurements should also be undertaken to run donation programs.

Sensitization to the mitochondrial pathway of apoptosis augments melanoma tumor cell responses to conventional chemotherapeutic regimens.

Metastatic malignant melanoma is highly resistant to chemotherapy, and the average survival rate is under 1 year. The only FDA-approved conventional chemotherapy (i.e., dacarbazine) targets melanoma tumor cells by inducing a form of cell death referred to as apoptosis. However, dacarbazine exhibits a response rate of ~5%, and combination chemotherapies consisting of cisplatin, vinblastine, and dacarbazine often offer little clinical advantage over dacarbazine alone. Apoptosis is governed by the BCL-2 family of proteins, which is comprised of anti-apoptotic and pro-apoptotic members. To determine if the anti-apoptotic BCL-2 repertoire established the cell death threshold and chemoresistance in melanoma, a novel treatment strategy was designed to inhibit the anti-apoptotic BCL-2 members with ABT-737. Using various melanoma model systems, we determined the affects of ABT-737 on sensitivity to dacarbazine-based regimens. Strikingly, ABT-737 re-sensitized melanoma cell lines to common chemotherapeutics leading to marked BIM-mediated apoptosis. Cellular features of the ABT-737 combination treatments were loss of proliferation, mitochondrial fragmentation, nuclear condensation, phosphatidylserine exposure, and decreased clonogenic survival. We also observed significant anti-tumor activity in an in vivo melanoma model system. Our data indicate that ABT-737 may be a beneficial adjuvant therapy to improve melanoma response rates when conventional chemotherapy is the only option.

Highly sensitive thermal conductivity measurements of suspended membranes (SiN and diamond) using a 3ω-Völklein method.

A suspended system for measuring the thermal properties of membranes is presented. The sensitive thermal measurement is based on the 3ω dynamic method coupled to a Völklein geometry. The device obtained using micro-machining processes allows the measurement of the in-plane thermal conductivity of a membrane with a sensitivity of less than 10 nW/K (+∕-5 × 10(-3) Wm(-1) K(-1) at room temperature) and a very high resolution (ΔK/K = 10(-3)). A transducer (heater/thermometer) centered on the membrane is used to create an oscillation of the heat flux and to measure the temperature oscillation at the third harmonic using a Wheatstone bridge set-up. Power as low as 0.1 nW has been measured at room temperature. The method has been applied to measure thermal properties of low stress silicon nitride and polycrystalline diamond membranes with thickness ranging from 100 nm to 400 nm. The thermal conductivity measured on the polycrystalline diamond membrane support a significant grain size effect on the thermal transport.

Dihalide and pseudohalide radical anions as oxidizing agents in nonaqueous solvents.

The dihalide and pseudohalide radical anions, strong one-electron oxidants, can be selectively generated in aqueous solutions by pulse radiolysis. Radiolysis of salts of the bulky 1-butyl-3-methylimidazolium cation with Cl(-), Br(-), SCN(-), and N(3)(-) anions allows efficient generation of the same species (Cl(2)(*-), Br(2)(*-), (SCN)(2)(*-), and N(6)(*-) radical anions) also in neat ionic liquids and in nonaqueous solvents with the addition of small amounts of the salt. The oxidative power of dichloride radical anion, lowered in the dichloromethane as compared to the aqueous solution, remains sufficient for oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD, (2)k = (2.0 +/- 0.1) x 10(9) M(-1) s(-1)), phenothiazine (PZ, (2)k = (2.1 +/- 0.1) x 10(9) M(-1) s(-1)), 10-methylphenothiazine (MPZ, (2)k = (9.3 +/- 0.1) x 10(7) M(-1) s(-1)), and 3-methylindole (scatole, SCT, (2)k = (6.8 +/- 0.1) x 10(7) M(-1) s(-1)). It diminishes on going to Br(2)(*-) (reacts only with TMPD, (2)k = (5.4 +/- 0.3) x 10(8) M(-1) s(-1), and PZ, (2)k = (4.8 +/- 0.3) x 10(8) M(-1) s(-1)) while (SCN)(2)(*-) and N(6)(*-) radical anions oxidize only TMPD, (2)k = (5.1 +/- 0.5) x 10(8) M(-1) s(-1) and (2)k approximately 10(8) M(-1) s(-1), respectively.

Dual EGFR and COX-2 inhibition as a novel approach to targeting head and neck squamous cell carcinoma.

Epidermal growth factor inhibition (EGFR) is emerging as an important treatment modality in several epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite some notable successes, less than 20% of patients respond to EGFR inhibition due to intrinsic and acquired resistance. Since EGFR inhibition is already used for lung, colorectal, breast and pancreas cancers in addition to HNSCC, overcoming treatment resistance would have a major impact on outcome. When the mechanisms of intrinsic resistance are identified, including mutations in the EGFR receptor, alternative therapeutic approaches should be employed. Mechanisms of acquired resistance that may be amenable to pharmacological therapies include dysregulation of EGFR degradation, constitutive activation of overlapping signal transduction pathways, especially cMET/HER3, the PI3K/Akt resistance pathway, angiogenesis and epithelial to mesenchymal transition. COX-2 is another promising target for HNSCC and preclinical data suggest that COX-2 inhibitors can affect most of the described acquired EGFR resistance pathways. Several combined EGFR and COX-2 inhibition trials have been completed and demonstrate promise for HNSCC. Combinatorial strategies of combined inhibition of EGFR and acquired resistance pathways in combination with radiation or chemotherapy are warranted.

Clinical evaluation of proinflammatory cytokine inhibitors (sTNFR I, sTNFR II, IL-1 ra), anti-inflammatory cytokines (IL-10, IL-13) and activation of neutrophils after burn-induced inflammation.

The study was aimed at evaluating the involvement of sTNFR I, sTNFR II, IL-1 ra, IL-10, IL-13 and reactive oxygen species (ROS) in systemic inflammatory response syndrome (SIRS) development in severely burned children and at assessing the prognostic value of the immunological markers studied. The study comprised 37 patients (17 burned children and 20 controls). Serum levels of the markers determined by means of ELISA and respiratory burst of neutrophils as well as p55 and p75 tumour necrosis factor-alpha (TNF-alpha) receptor expression using flow cytometry were evaluated twice. The burned children presented significantly higher levels of IL-10 and cytokine inhibitors within the first 6-24 h after injury compared with controls (P < 0.05). The decreased oxygen metabolism of neutrophils and increased TNF-alpha receptor expression were found on admission. Moreover, a significant decrease in initially high sTNFR I, sTNFR II, IL-1 ra, IL-10, IL-13 concentrations (P < 0.05) and reduced expression of TNF-alpha receptors (P < 0.05) were observed after burn therapy, whereas ROS generation evidently augmented (P < 0.05). Four of our children who developed hypovolaemic shock revealed a significantly lower ROS generation and higher concentrations of soluble TNF-alpha receptors and IL-1 ra together with IL-10, IL-13 compared with children with good outcome (P < 0.05). Our results revealed the involvement of both ROS, soluble TNF-alpha receptors and IL-1 ra in the development of SIRS in burned children; their monitoring allows for an assessment of the systemic inflammatory reaction activity. The neutrophil BURSTTEST and IL-1 ra might have been clinically helpful markers of SIRS prognosis.

Heat shock response in gastrointestinal tract.

Heat shock response is one of the defense mechanisms common to eukaryotic and prokaryotic cells. The highly conserved and ubiquitous heat shock proteins (HSPs) are essential for cell survival during stress. Stress tolerance, i.e., adaptation of cells to stress conditions, is a characteristic feature of heat shock response. The lumen of the gastrointestinal tract is an external environment to the body. Epithelium of the digestive tract is exposed to various stress factors inducing the heat shock response, e.g., bacteria and their toxins, food borne chemical compounds, drugs and diet deficiencies. Other factors like plant lecitins, glutamine or short fatty acids are mild stressors and can modulate the heat shock response in cells. All these factors are presumed to influence the normal microflora that is an integral part of the digestive tract. This review is focused on the induction/modulation of heat shock proteins expression in the epithelium of the gastrointestinal tract by various factors, on the protective role of HSPs and mechanisms leading to stress protection inside the gut. Heat shock response is one of the key mechanisms of maintenance of gastrointestinal tract homeostasis. It is involved in pathogenic bacteria adaptation to life in the digestive tract, especially in colony formation and in their role in infectious processes.

The Vibrio harveyi GTPase CgtAV is essential and is associated with the 50S ribosomal subunit.

It was previously reported that unlike the other obg/cgtA GTPases, the Vibrio harveyi cgtAV is not essential. Here we show that cgtAV was not disrupted in these studies and is, in fact, essential for viability. Depletion of CgtAV did not result in cell elongation. CgtAV is associated with the large ribosomal particle. In light of our results, we predict that the V. harveyi CgtAV protein plays a similar essential role to that seen for Obg/CgtA proteins in other bacteria.

Biochemical properties of the Vibrio harveyi CgtAV GTPase.

Bacteria encode a number of relatively poorly characterized GTPases, including the essential, ribosome-associated Obg/CgtA proteins. In contrast to Ras-like proteins, it appears that the Obg/CgtA proteins bind guanine nucleotides with modest affinity and hydrolyze GTP relatively slowly. We show here that the Vibrio harveyi CgtA(V) exchanges guanine nucleotides rapidly and has a modest affinity for nucleotides, suggesting that these features are a universal property of the Obg/CgtA family. Interestingly, CgtA(V) possesses a significantly more rapid GTP hydrolysis rate than is typical of other family members, perhaps reflecting the diversity and specificity of bacterial ecological niches.

Thermoresponsive polymers as promising new materials for local radiotherapy.

We describe a novel thermoresponsive polymeric drug delivery system based on poly(N-isopropylacrylamide) with isotopically labellable end groups [l-tyrosinamide or diethylenetriaminepentaacetic acid (DTPA)] designed for local radiotherapy. The polymers are readily soluble in isotonic aqueous sodium chloride at room temperature and the phase separation is complete at body temperature as proved by DSC measurements. Sufficent binding capacity for radionuclides and chemical stability are demonstrated on 125I and 90Y-labelled polymers.

Genotoxicity of streptozotocin in normal and cancer cells and its modulation by free radical scavengers.

Streptozotocin (STZ) is an antibiotic which can be used to induce diabetes in experimental animals in order to have an insight into pathogenesis of this disease. To use STZ as a diabetogenic substance, its molecular mode of action should be elucidated. Using the alkaline comet assay, we showed that STZ at concentrations in the range 0.01-100 micromol/L induced DNA damage in normal human lymphocytes and HeLa cancer cells in a dose-dependent manner. Lymphocytes were able to remove damage to their DNA within a 30-min repair incubation, whereas HeLa cells completed the repair in 60 min. Vitamins C and E at 10 and 50 micromol/L diminished the extent of DNA damage induced by 50 micromol/L STZ. Pretreatment of the lymphocytes with the nitrone spin trap, alpha-(4-pyridil-1-oxide)-N-tert-butylnitrone (POBN) or ebselen, which mimics glutathione peroxidase, or pyrrolidine dithiocarbamate (PDTC) reduced the extent of DNA damage evoked by STZ. The cells exposed to STZ and treated with endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. These results suggest that free radicals may be involved in the formation of DNA lesions induced by streptozotocin. The drug can also alkylate DNA bases. This broad range of DNA damage induced by STZ indicates that the drug may seriously affect genomic stability in normal and pathological cells.

Selection and validation of differentially expressed genes in head and neck cancer.

We applied a robust combinatorial (multi-test) approach to microarray data to identify genes consistently up- or down-regulated in head and neck squamous cell carcinoma (HNSCC). RNA was extracted from 22 paired samples of HNSCC and normal tissue from the same donors and hybridized to the Affymetrix U95A chip. Forty-two differentially expressed probe sets (representing 38 genes and one expressed sequence tag) satisfied all statistical tests of significance and were selected for further validation. Selected probe sets were validated by hierarchical clustering, multiple probe set concordance, and target-subunit agreement. In addition, real-time PCR analysis of 8 representative (randomly selected from 38) genes performed on both microarray-tested and independently obtained samples correlated well with the microarray data. The genes identified and validated by this method were in comparatively good agreement with other rigorous HNSCC microarray studies. From this study, we conclude that combinatorial analysis of microarray data is a promising technique for identifying differentially expressed genes with few false positives.