Cellular proliferation is a major determinant of the biologic behavior of breast cancer. Prognosis is apparently best indicated by the percentage of cells in S through M phases of the cell cycle. Measurement of the Ki-S2 labeling index of a tumor sample may improve a clinician's ability to make an accurate prognosis and to identify patients with a low risk of recurrence who may not need adjuvant therapy.
Protein fractionation by means of microfiltration (MF) is significantly affected by fouling, especially when spiral-wound membranes (SWMs) are used. We investigated the influence of the mode of transmembrane pressure (ΔpTM) increase to target level and the deposit layer pressure history on the filtration performance during skim milk MF at temperatures of 10 °C and 50 °C. Two filtration protocols were established: No. 1: ΔpTM was set directly to various target values. No. 2: Starting from a low ΔpTM, we increased and subsequently decreased ΔpTM stepwise. The comparison of both protocols tested the effect of the mode of ΔpTM increase to target level. The latter protocol alone tested the effect of the deposit layer history with regard to the ΔpTM. As expected, flux and protein permeation were both found to be functions of the ΔpTM. Further, both measures were independent of the filtration protocol as long as ΔpTM was held at a constant level or, as part of protocol No. 2, ΔpTM was increased. Thus, we can state that the mode of ΔpTM increase to target level does not affect filtration performance in SWM. We found that after completion of a full cycle of stepping ΔpTM up from 0.5 bar to 3.0 bar and back down, flux and deposit layer resistance were not affected by the deposit layer history at 10 °C, but they were at 50 °C. Protein permeation, however, was lower for both 10 °C and 50 °C, when the ΔpTM cycle was completed. The processing history had a significant impact on filtration performance due to remaining structural compression effects in the deposited layer, which occur most notably at higher temperatures. Furthermore, temperatures of 50 °C lead to deposit layer aging, which is probably due to an enhanced crosslinking of particles in the deposit layer. Apart from that, we could show that fouling resistance does not directly correlate with protein permeation during skim milk MF using SWM.
Purpose: The monoclonal antibody Ki-A10 (IgG1) generated after immunization of mice with Hodgkin's lymphoma cell line L428 detects a nuclear antigen in human tissues with a restricted distribution pattern similar to cancer/testis antigens.The aim of this study was to characterize the antigen and to determine the expression profile in Hodgkin's lymphoma. Experimental Design: The half-life and phosphorylation of the antigen were determined by radiolabeling. The antigen was characterized by immunopurification and sequencing. Demethylation of genes is used to induce cancer/testis antigens. Ki-A10-negative cells were treated with 5-aza-2 ¶-deoxycytidine. The Ki-A10 expression in paraffin-embedded tumors was determined immunohistochemically. Results: Immunopurification of the 25/22-kDa antigen and sequencing revealed a peptide of 14 amino acids corresponding to the gene product of the newly described gene family MGC27005, located on chromosome Xq26.3, now termed CT45. CT45 is significantly phosphorylated and down-regulated during mitosis. Demethylation of CT45-negative HeLa cells and stimulated peripheral blood lymphocytes induced CT45 expression. Except testis, immunohistochemical stainings of normal tissues, reactive lymphoid lesions, and most malignant tumors were negative. In comparison, 54 of 99 (55%) samples from pediatric and adolescent Hodgkin's lymphoma patients enrolled in the multicenter trial HD-95 stained Ki-A10 positive. Ki-A10 expression correlated with histologic subtypes (nodular sclerosis Hodgkin's lymphoma 68% versus mixed cellularity Hodgkin's lymphoma 40% versus nodular lymphocyte predominant Hodgkin's lymphoma 9%; P < 0.001).Conclusions: Ki-A10 is the first monoclonal antibody that detects CT45. As benign lymphoid lesions did not express CT45, the use of Ki-A10 antibody will facilitate the discrimination of Hodgkin's lymphoma from reactive lymphadenopathies.
BackgroundDue to their restricted expression in male germ cells and certain tumors, cancer/testis (CT) antigens are regarded as promising targets for tumor therapy. CT45 is a recently identified nuclear CT antigen that was associated with a severe disease score in Hodgkin’s lymphoma and poor prognosis in multiple myeloma. As for many CT antigens, the biological function of CT45 in developing germ cells and in tumor cells is largely unknown.MethodsCT45 expression was down-regulated in CT45-positive Hodgkin’s lymphoma (L428), fibrosarcoma (HT1080) and myeloma (U266B1) cells using RNA interference. An efficient CT45 knock-down was confirmed by immunofluorescence staining and/or Western blotting. These cellular systems allowed us to analyze the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells.ResultsReduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, we observed alterations in cell adherence, morphology and migration/invasion after CT45 down-regulation. Significant changes in the distribution of cytoskeleton-associated proteins were detected by confocal imaging. Changes in cell adherence were recorded in real-time using the xCelligence system with control and siRNA-treated cells. Altered migratory and invasive capacity of CT45 siRNA-treated cells were visualized in 3D migration and invasion assays. Moreover, we found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility.ConclusionsProviding first evidence of a cell biological function of CT45, we suggest that this cancer/testis antigen is involved in the modulation of cell morphology, cell adherence and cell motility. Enhanced motility and/or invasiveness of CT45-positive cells could contribute to the more severe disease progression that is correlated to CT45-positivity in several malignancies.
The use of bioactive bovine milk immunoglobulins (Ig) has been found to be an alternative treatment for certain human gastrointestinal diseases. Some methodologies have been developed with bovine colostrum. These are considered in laboratory scale and are bound to high cost and limited availability of the raw material. The main challenge remains in obtaining high amounts of active IgG from an available source as mature cow milk by the means of industrial processes. Microfiltration (MF) was chosen as a process variant, which enables a gentle and effective concentration of the Ig fractions (ca. 0.06% in raw milk) while reducing casein and lactose at the same time. Different microfiltration membranes (ceramic standard and gradient), pore sizes (0.14–0.8 µm), transmembrane pressures (0.5–2.5 bar), and temperatures (10, 50 °C) were investigated. The transmission of immunoglobulin G (IgG) and casein during the filtration of raw skim milk (<0.1% fat) was evaluated during batch filtration using a single channel pilot plant. The transmission levels of IgG (~160 kDa) were measured to be at the same level as the reference major whey protein β-Lg (~18 kDa) at all evaluated pore sizes and process parameters despite the large difference in molecular mass of both fractions. Ceramic gradient membranes with a pore sizes of 0.14 µm showed IgG-transmission rates between 45% to 65% while reducing the casein fraction below 1% in the permeates. Contrary to the expectations, a lower pore size of 0.14 µm yielded fluxes up to 35% higher than 0.2 µm MF membranes. It was found that low transmembrane pressures benefit the Ig transmission. Upscaling the presented results to a continuous MF membrane process offers new possibilities for the production of immunoglobulin enriched supplements with well-known processing equipment for large scale milk protein fractionation.
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