Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Physiological routes from intra-uterine seminal contents to advancement of ovulation

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.     

Monitoring signal transduction in cancer: tyrosine kinase gene expression profiling.

Abnormal expression of tyrosine kinase (TK) genes is common in tumors, in which it is believed to alter cell growth and response to external stimuli such as growth factors and hormones. Although the etiology and pathogenesis of carcinomas of the thyroid or breast remain unclear, there is evidence that the expression of TK genes, such as receptor tyrosine kinases, or mitogen-activated protein kinases, is dysregulated in these tumors, and that overexpression of particular TK genes due to gene amplification, changes in gene regulation, or structural alterations leads to oncogenic transformation of epithelial cells. We developed a rapid scheme to measure semiquantitatively the expression levels of 50-100 TK genes. Our assay is based on RT-PCR with mixed based primers that anneal to conserved regions in the catalytic domain of TK genes to generate gene-specific fragments. PCR products are then labeled by random priming and hybridized to DNA microarrays carrying known TK gene targets. Inclusion of differently labeled fragments from reference or normal cells allows identification of TK genes that show altered expression levels during malignant transformation or tumor progression. Examples demonstrate how this innovative assay might help to define new markers for tumor progression and potential targets for disease intervention. (J Histochem Cytochem 49:673-674, 2001)     

Early senescence and production of senescence-associated cytokines are major determinants of radioresistance in head-and-neck squamous cell carcinoma

Resistance against radio(chemo)therapy-induced cell death is a major determinant of oncological treatment failure and remains a perpetual clinical challenge. The underlying mechanisms are manifold and demand for comprehensive, cancer entity- and subtype-specific examination. In the present study, resistance against radiotherapy was systematically assessed in a panel of human head-and-neck squamous cell carcinoma (HNSCC) cell lines and xenotransplants derived thereof with the overarching aim to extract master regulators and potential candidates for mechanism-based pharmacological targeting. Clonogenic survival data were integrated with molecular and functional data on DNA damage repair and different cell fate decisions. A positive correlation between radioresistance and early induction of HNSCC cell senescence accompanied by NF-κB-dependent production of distinct senescence-associated cytokines, particularly ligands of the CXCR2 chemokine receptor, was identified. Time-lapse microscopy and medium transfer experiments disclosed the non-cell autonomous, paracrine nature of these mechanisms, and pharmacological interference with senescence-associated cytokine production by the NF-κB inhibitor metformin significantly improved radiotherapeutic performance in vitro and in vivo. With regard to clinical relevance, retrospective analyses of TCGA HNSCC data and an in-house HNSCC cohort revealed that elevated expression of CXCR2 and/or its ligands are associated with impaired treatment outcome. Collectively, our study identifies radiation-induced tumor cell senescence and the NF-κB-dependent production of distinct senescence-associated cytokines as critical drivers of radioresistance in HNSCC whose therapeutic targeting in the context of multi-modality treatment approaches should be further examined and may be of particular interest for the subgroup of patients with elevated expression of the CXCR2/ligand axis.Subject terms: Radiotherapy, Head and neck cancer, Senescence, Tumour heterogeneity

     

A rapid ex vivo tissue model for optimising drug detection and ionisation in MALDI imaging studies

The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.     

Heterogeneous chromosomal aberrations in intraductal breast lesions adjacent to invasive carcinoma.

There is evidence that breast cancer is a heterogeneous disease phenotypically as well as molecular biologically. So far, heterogeneity on the molecular biological level has not been investigated in potential precursor lesions, such as ductal hyperplasia (DH) and ductal carcinoma in situ (DCIS). In this study we applied comparative genomic hybridization (CGH) to formalin-fixed, paraffin-embedded breast tissue with DH and DCIS, adjacent to invasive ductal carcinoma (IDC), to screen these potential precursor lesions for whole genomic chromosomal imbalances. Laser-microdissection was used to select pure cell populations from the sections. Isolated DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and further processed for CGH analysis. Investigating multiple samples (n = 25) from four patients we found an average of 5.6 +/- 0.9 (mean +/- SEM) chromosomal imbalances already present in DH. In the twelve DCIS lesions an average of 10.8 (+/- 0.9) aberrations was identified with 14.8 (+/- 0.8) aberrations in the four adjacent IDC lesions. The increasing number of chromosomal changes in parallel with the histopathological sequence corroborate the hypothesis, that the carcinomas may have developed through a sequential progression from normal to proliferative epithelium and eventually into carcinoma. However, heterogeneous results were identified in the multiple samples per entity from the same patient, demonstrated mainly in the DCIS samples in the chromosomal regions 6p, 9p, 11q, 16p and 17q, in the DH samples by 3p, 16p and 17q. This heterogeneous findings were most pronounced within the DH and was less in the DCIS and IDC samples. The only aberration consistently found in all samples-even in all DH sample-was amplification of the 20q13 region. Our results demonstrate, that the applied combination of laser-microdissection, DOP-PCR and CGH, may serve to analyse breast carcinogenesis pathways in suitable histological material. However, so far, it is unclear how to handle heterogeneous results and these make identification of relevant changes more difficult. Setting a threshold and evaluating only those chromosomal changes which are present in a majority of samples may be one possibility. This involves however, the risk that infrequent but possibly significant aberrations may be missed. Figures on http://www.esacp.org/acp/2000/20-1/aubele. htm.     

Doses of Ukrainian female clean-up workers with diagnosed breast cancer

The Chernobyl reactor accident in 1986 has caused significant exposure to ionizing radiation of the Ukrainian population, in particular clean-up workers and evacuees from the exclusion zones. A study aiming at the discovery of radiation markers of the breast cancer was conducted from 2008 to 2015 within a collaborative project by HZM, LMU, and NRCRM. In this study, post-Chernobyl breast cancer cases both in radiation-exposed female patients diagnosed at age less than 60 from 1992 to 2014 and in non-exposed controls matched for residency, tumor type, age at diagnosis, TNM classification as well as tumor grading were investigated for molecular changes with special emphasis to copy number alterations and miRNA profiles. Cancer registry and clinical archive data were used to identify 435 breast cancer patients among female clean-up workers and 14 among evacuees from highly contaminated territories as candidates for the study. Of these, 129 breast cancer patients fit study inclusion criteria and were traced for individual reconstruction of the target organ (breast) doses. The doses were estimated for 71 exposed cases (clean-up workers and evacuees from which biomaterial was available for molecular studies and who agreed to participate in a dosimetric interview) by the use of the well-established RADRUE method, which was adjusted specifically for the assessment of breast doses. The results of 58 female clean-up workers showed a large inter-individual variability of doses in a range of about five orders of magnitude: from 0.03 to 929 mGy, with median of 5.8 mGy. The study provides the first quantitative estimate of exposures received by female clean-up workers, which represent a limited but very important group of population affected by the Chernobyl accident. The doses of 13 women evacuated after the accident who did not take part in the clean-up activities (from 4 to 45 mGy with median of 19 mGy) are in line with the previous estimates for the evacuees from Pripyat and the 30-km zone.     

Differential staining of human and murine chromatin in situ by hybridization with species-specific satellite DNA probes.

Human and murine chromatin was differentially labeled by hybridization with DNA probes that bind to species-specific satellite DNA. The targets for in situ hybridization were the mouse-specific major or gamma satellite DNA and the human alpha satellite DNA. These sequences typically are localized at or near the chromosome centromeres, and remain their tight localization throughout the cell cycle. DNA probes were synthesized in vitro by primer directed DNA amplification using the polymerase chain reaction. In typical applications like the differentiation of cells derived from chimeric animals or the characterization of chromosomes in somatic cell hybrids, the two DNA probes are differently labeled and detected using label-specific reagents that fluoresce at different wavelengths. The rapid technique for chromatin discrimination described here combines high specificity with unprecedented signal intensity.