Left-right difference in fetal liver oxygenation during hypoxia estimated by BOLD MRI in a fetal sheep model.

OBJECTIVE: The purpose of this study was to measure differences in oxygenation between the left and right sides of the fetal liver during varying oxygenation levels. METHODS: Eight ewes carrying singleton fetuses at gestational age 125 days (term, 145 days) were included in the study. Under anesthesia the ewes were ventilated with gas containing different levels of oxygen, thereby subjecting the fetuses to hyperoxia (mean ± SD maternal arterial partial pressure of oxygen (pO2), 23.2 ± 8.2 kPa) and hypoxia (mean maternal arterial pO2, 7.1 ± 0.5 kPa). Changes in oxygenation within the fetal liver were assessed by blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI). RESULTS: During hyperoxia there was no difference between the BOLD signal in the left and right sides of the fetal liver; mean change in BOLD (ΔBOLD)(hyperox), -0.9 ± 3.7%. During hypoxia, however, the decrease in the BOLD signal was more pronounced in the right side as compared with the left side, thereby creating a significant increase in the left-right difference in the BOLD signal; mean ΔBOLD(hypox), 5.2 ± 2.2% (P = 0.002, paired t-test). The left-right difference was directly proportional to the degree of hypoxia (R2 = 0.86, P = 0.007). CONCLUSIONS: To our knowledge, this is the first study demonstrating differences in oxygenation between the left and right sides of the fetal liver during hypoxia, a difference that can be explained by increased ductus venosus shunting. Thus, the BOLD MRI technique is a promising non-invasive tool that might be useful for the future monitoring of the human fetus.

HGF is released from buccal fibroblasts after smokeless tobacco stimulation.

To investigate the effect of smokeless tobacco (ST) on (1) HGF, KGF and GM-CSF expression by buccal fibroblasts and (2) on keratinocyte and fibroblast proliferation. Buccal fibroblasts were stimulated with different concentrations of ST extracts in a double dilution from 0.50% w/v to 0.03% w/v. Supernatant was collected after 24/48/72/96 h and assayed for HGF, KGF, and GM-CSF by ELISA. The amount of RNA was used as an indicator of fibroblast cell number. Buccal epithelial cell proliferation was determined by CyQUANT proliferation assay. The amount of HGF and KGF in the supernatant was dependent on exposure time and on concentration of the tobacco extract. High concentration increased production of HGF 4-fold. KGF production was doubled when high concentration of tobacco was used, low concentration did not stimulate cells. GM-CSF production was low in both stimulated and non-stimulated cells. Keratinocytes and fibroblasts showed no increase in proliferation after stimulation with increased concentrations of ST. The results suggest that HGF and KGF may play an important role as a paracrine growth factor in epithelial hyperplasia in ST lesions.

De novo identification of cell-type specific antibody-antigen pairs by phage display subtraction. Isolation of a human single chain antibody fragment against human keratin 14.

The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly non-differentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.

p53 accumulation associated with bcl-2, the proliferation marker MIB-1 and survival in patients with prostate cancer subjected to watchful waiting.

PURPOSE: We describe the association of p53 nuclear protein accumulation with bcl-2 expression, tumor cell proliferation and clinical outcome in a prostate cancer population undergoing watchful waiting. MATERIALS AND METHODS: Immunohistochemical staining for p53 was semiquantitatively scored in archival formalin fixed, paraffin embedded tumor tissue obtained at diagnosis in 221 patients with prostate cancer. At a median of 15 years followup was nearly complete. Eventually 57% of the patients died of prostate cancer. RESULTS: p53 Immunohistochemical staining was heterogeneous but in all cases at least clusters of tumor cells had nuclear staining for p53. The percent of p53 immunoreactive tumor cells was scored as 0 to 4+ in p53 positive hot spots. p53 immunoreactivity correlated with clinical stage and histopathological grade (p = 0.003 and 0.009, respectively). When dichotomized into low (0% to 50%) and high (51% to 100%) immunoreactivity groups of 40 and 181 patients, respectively, p53 accumulation was significantly associated with disease specific survival in the study population overall (p <0. 0001) and in the 125 with clinically localized disease (p = 0.0002). p53 Immunoreactivity was significantly (p <0.001) associated with the proliferation marker MIB-1 (median value 10.3, range 0 to 46.1) but insignificantly (p = 0.8) correlated with bcl-2 expression (52% positive). However, patients with combined favorable MIB-1 and bcl-2 status were stratified into significant (p = 0.02) prognostic groups by p53 immunohistochemical status. Multivariate analysis revealed that p53 immunoreactivity was a significant prognostic factor in patients with clinically localized prostate cancer (p <0.0001). CONCLUSIONS: p53 Nuclear protein accumulation detected by immunohistochemical study was an independent adverse prognostic factor in patients with prostate cancer undergoing watchful waiting.

Blood-group-related carbohydrates are expressed in organotypic cultures of human skin and oral mucosa.

Cellular maturation and migration are usually associated with changes in cell-surface carbohydrates, but the relationship between these changes and cell behaviour is at present largely unknown. To investigate whether an organotypic culture system can be used as an in vitro model to study the function of cell-surface carbohydrates, we established organotypic cultures of skin and buccal mucosa. In these cultures, keratinocytes are grown at the air-liquid interface on a supporting matrix consisting of homologous fibroblasts embedded in a collagen type I gel. We examined the expression of blood-group-related carbohydrate structures, including Lewis x, sialylated Lewis x, Lewis y, Lewis a, and Lewis b, on the surface of epithelial cells in the cultures. We compared the results with the expression of more well-established markers, including cytokeratins, integrins, bullous pemphigoid antigen and laminin, in the same cultures. The organotypic skin and oral mucosa cultures showed a histological differentiation pattern analogous to that of normal skin and buccal mucosa, and a tissue-specific expression of carbohydrate structures and cytokeratins. However, both types of organotypic cultures also expressed markers which are normally seen during wound healing, including Lewis y, cytokeratin 16, and cytokeratin 19. We conclude that the organotypic cultures of oral mucosa and skin are suitable models for future studies of the function of cell-surface carbohydrates, although the expression of wound healing markers has to be taken into consideration.

Relationship between tumour cell in vitro radiosensitivity and clinical outcome after curative radiotherapy for squamous cell carcinoma of the head and neck.

BACKGROUND AND PURPOSE: Clinically, it is recognized that individual tumours respond differently to radiation treatment. Variation in tumour cell radiosensitivity is believed to be an important underlying factor. In the current study, cellular in vitro radiosensitivity was estimated as the fraction of surviving cells after a radiation dose of 2 Gy (SF2) and related to clinical outcome after curative radiotherapy. PATIENTS AND METHODS: Thirty-eight patients with squamous cell carcinoma of the head and neck were treated with curative radiotherapy alone. Pre-treatment biopsies were disaggregated to form a single-cell suspension and cells were cultured in the modified Courtenay-Mills soft agar clonogenic assay. Directly from this assay and with no respect to cell type, overall SF2 was assessed. By collecting the obtained colonies on a preparation slide using a colony-filter technique, and with immunocytochemical staining, it was possible to measure the surviving fraction of tumour cells selectively as tumour cell SF2. RESULTS: Experimentally, a broad inter-tumour variation was found for both tumour cell SF2 and overall SF2. Using weighted linear regression, it was demonstrated that tumour cell SF2 and overall SF2 were two independent measures of tumour radiosensitivity. In general, the measures of tumour radiosensitivity were independent of patient sex and age, T- and N-category, disease stage, tumour size and plating efficiency. Among the 38 patients grouped in loco-regional failures and patients with loco-regional control, respectively, sex, age, total radiation dose, overall treatment time and tumour grade were equally distributed. Advanced stage, lymph node involvement and tumour size correlated statistically significantly with poor loco-regional control. Neither tumour cell SF2, overall SF2, nor plating efficiency predicted loco-regional tumour control probability. In a multivariate analysis with respect to the risk of loco-regional tumour failure, only disease stage yielded independent prognostic significance. This significance suggests that this patient sample was representative for the patient population with head and neck cancer. CONCLUSION: In 38 patients with squamous cell carcinoma of the head and neck, the estimated tumour radiosensitivities were not statistically related to clinical outcome after curative radiotherapy alone.

In vitro radiosensitivity of tumour cells and fibroblasts derived from head and neck carcinomas: mutual relationship and correlation with clinical data.

The aim was to characterize the variation in the cellular in vitro radiosensitivities in squamous cell carcinomas of the head and neck, and to test for a possible correlation between different measures of radiosensitivity and the clinical and histopathological data. Cellular in vitro radiosensitivities were assessed in tumour biopsies from 71 patients using the modified Courtenay-Mills soft agar clonogenic assay combined with an immunocytochemical analysis. Radiosensitivity was quantified as the surviving fraction after a radiation dose of 2 Gy irrespective of cell type (overall SF2), or based on identification of cell type (tumour cell SF2, fibroblast SF2). Sixty-three biopsies were from primary tumours, and eight were from recurrences. Overall plating efficiency ranged from 0.005 to 1.60% with a median of 0.052%. The majority of the colonies obtained from the biopsies were fibroblast marker-positive; the proportion of tumour marker-positive colonies ranged from 1 to 88% with a median of 15%. The median overall SF2 was 0.47 (range 0.24-0.96), the median tumour cell SF2 was 0.50 (range 0.11-1.0) and the median fibroblast SF2 was 0.49 (range 0.24-1.0). Comparing data from independent experiments, the overall SF2 was significantly correlated with the SF2 of fibroblasts (2P = 0.006) but not with the tumour cell SF2. The tumour cell and fibroblast radiosensitivities measured in the same individuals were not correlated (r= 0.06, 95% CI [-0.19, 0.30]):This finding seems to preclude a strong correlation between the radiosensitivity of tumour cells and fibroblasts. Concerning the clinical characteristics, neither of the measures of tumour radiosensitivity was correlated with T- and N-category, stage, tumour size, sex and age. However, the tumour cell radiosensitivity decreased with increasing grade of histopathological differentiation (2P = 0.012). The same tendency was found in two independent analyses of the same patient material. This correlation was not significant in case of the overall SF2 or the fibroblast SF2.

Altered expression of epithelial cell surface glycoconjugates and intermediate filaments at the margins of mucosal wounds.

Alterations in cell to cell adhesion are necessary to enable the type of cell movements that are associated with epithelial wound healing and malignant invasion. Several studies of transformed cells have related epithelial cell movement to changes in the cell surface expression of the carbohydrate structures represented by the ABO blood group antigens and, in particular, by Lewis antigens and their biosynthetic precursors. To study further the relationship between cell surface carbohydrates and keratinocyte cell movement, experimental wounds were created in human oral mucosa and examined by immunohistochemical methods for their expression of selected cytokeratins (K5, K16, K19), basement membrane components (laminin alpha5 and gamma2-chains, BP180, collagen IV and collagen VII), and blood group antigen precursor structures Le(x), sialosyl-Le(x), Le(y), H antigen, N-acetyllactosamine, and sialosyl-T antigen. The changes induced by wounding in the expression of collagen IV, laminin gamma2-chain (laminin-5), and laminin alpha5-chain were similar to those found in skin wounds and served to define the region of epithelial movement. This region was found to show a marked increase in staining for both Lewis antigen Y (Le(y)) and H blood group antigen, and decreased staining of Le(x), thus indicating an upregulation in wounded epithelium of the fucosyltransferases responsible for the synthesis of the H antigen. The changes in carbohydrate expression extended beyond the wound margin into the nonwounded epithelium, a pattern of expression similar to K16, which was also strongly upregulated in both the outgrowth and the adjacent nonwounded epithelium. These findings provide further support for an influence of such carbohydrate structures on the migratory behavior of epithelial cells.

Radiosensitivity of normal fibroblasts from breast cancer patients assessed by the micronucleus and colony assays.

PURPOSE: To investigate whether radiation-induced micronucleus formation as expressed by the cytochalasin-blocked Mn-assay correlates with cellular radiosensitivity measured by a colony assay in primary fibroblast cultures from cancer patients. MATERIALS AND METHODS: Studies were made on skin fibroblasts from 36 breast cancer patients. The micronucleus assay was performed using treatment with cytochalasin-B to create binucleate cells. Response was scored in terms of the percentage of binucleate cells with micronuclei, also as the number of micronuclei per binucleate cell. The data were related to previously published results of cell survival measurements on these cell lines. RESULTS: Neither endpoint for micronucleus formation showed a correlation with radiosensitivity by the colony assay. The fraction of fibroblasts that reach mitosis without micronuclei also failed to correlate with cell survival. CONCLUSIONS: Among these primary fibroblast cell lines radiation-induced micronucleus formation was not associated with radiosensitivity as measured by a colony assay.

Glycerol diversifies phage repertoire selections and lowers non-specific phage absorption.

Using a semi-synthetic phage displayed antibody repertoire, isoform-specific and cross-reactive phage-antibodies to eukaryotic elongation factor 1A (eEF1A) have been selected. Enrichment of specific antibodies was found to depend on the presence of glycerol. Further selections against lactate dehydrogenase (LDH) revealed that the dominance of a phage-antibody clone to LDH was inhibited by glycerol, a notable feature for selection strategies where a broad variety of binding clones is desired. The impact of glycerol in distinct steps of the selection protocol was examined and glycerol found to affect certain antibody-antigen interactions. Furthermore, the nonspecific phage binding was lowered by three orders of magnitude at a 20% (v/v) glycerol concentration.

Immunohistochemical BCL-2 and Ki-67 expression predict survival in prostate cancer patients followed expectantly.

The prognostic value of BCL-2 expression, solely and combined with Ki-67 expression, was determined in prostate cancer patients followed expectantly. Furthermore, associations with well established prognostic markers were tested. Formalin fixed, paraffin-embedded tumour tissue obtained at diagnosis was immunohistochemically investigated in 221 patients with a 15 y median follow-up time. BCL-2 protein was expressed in 114 (52%) tumours and was significantly associated with tumour stage (P=0.01). The prognostic value of BCL-2 expression was significant, using both disease-specific (P=0.0015) and overall survival (P=0.005) as endpoint. Patients with a combined BCL-2 negative/Ki-67 'low' tumour had the most favourable prognosis. This combined BCL-2/Ki-67 variable was of independently prognostic value in both the entire population (P=0.0001) and in the clinically localized subpopulation (P=0.035).

Characterization and radiosensitivity of fibroblasts derived from squamous cell carcinomas of the head and neck, and the surrounding oral mucosa.

Recently, extensive stromal fibroblast contamination has been reported in the modified Courtenay-Mills soft agar clonogenic assay for cellular in vitro radiosensitivity in tumour biopsies. The aim of the present study was to evaluate the hypothesis that an immunocytochemical analysis added to the modified Courtenay-Mills soft agar clonogenic assay provides a measure of both fibroblast and tumour cell radiosensitivity. Therefore, fibroblasts derived from squamous cell carcinomas of the head and neck, and from the surrounding oral mucosa were compared for immunocytochemistry, DNA ploidy, plating efficiency and surviving fraction of cells after a radiation dose of 2 Gy. The results of our study suggest that the stromal fibroblasts derived from tumour biopsies are representative of normal fibroblasts with respect to the characteristics examined using mucosal fibroblasts as normal controls.

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts.

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa, from gingiva, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal nature of fibroblasts. Cells were first cultured in DMEM with 0.5% fetal calf serum (FCS) and then incubated for 8 h, and 72 h in fresh DMEM with 10% FCS. Total RNA was isolated and used for Northern blotting with a P32-labeled KGF cDNA probe. Total RNA from cultured keratinocytes was used as negative controls. KGF mRNA was found in all cultured fibroblasts. Upon addition of 10% FCS to the cell cultures, an increase in KGF mRNA levels was noticed especially after 72 h. Furthermore, RT-PCR analysis of material scraped from the tooth root surface indicated the presence of KGF mRNA even in noncultured periodontal ligament cells.

Retrieval of phage displayed scFv fragments using direct bacterial elution.

Phage displayed repertoires of antibody fragments, either single chain Fv (scFv) or Fab, have become a real alternative to traditional hybridoma technology in the generation of monoclonal antibodies. The steps usually taken in the selection from such repertoires were analysed and the necessity of chemical elution of bound phage-Abs and precipitation of amplified phage particles questioned. By using a semi-synthetic scFv library as a source, phage antibodies recognising a panel of seven antigens were isolated utilising direct bacterial elution of bound phage. Selections against two antigens were subsequently performed with bacterial or chemical elution in parallel and the resulting pools of phage antibodies compared. It is demonstrated that direct bacterial elution can be used when selecting from phage displayed antibody repertoires but that the enrichment of high affinity binders might be jeopardised. In addition, a simplified and more rapid scheme for amplification and use of phage displayed repertoires is described.

A model phage display subtraction method with potential for analysis of differential gene expression.

In order to establish a subtractive procedure that makes it possible to enrich selectively phage displayed antibodies directed against proteins constituting a difference between two populations of cells, a competitive selection strategy utilising two solid phases was developed and tested. Antibodies recognising a defined difference between two otherwise identical protein mixtures were isolated and their specificity confirmed. To test further the efficacy of selection inhibition during the competitive selections, selections towards a total cell extract were performed with and without competition from the same extract. An analysis of the resulting phage antibodies confirmed the subtractive nature of the system described.