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There is little information concerning the fractionation by microfiltration (MF) of casein micelles and immunoglobulins plus other minor whey proteins with ceramic gradient membranes. The order of transmission was α-lactalbumin (α-La, 2.3 nm)> β-lactoglobulin (β-Lg; 4.2 nm)>IgG (10.7 nm)> lactoperoxidase (8.2 nm)> IgA (18.1 nm)> IgM (23.8 nm)> lactoferrine > blood serum albumin (7.8 nm), irrespective of the applied transmembrane pressure (0.6–3 bar) and equal to 55% > 50% > 47% > 41%>39% > 32% > 22% > 19%. Including preconcentration, it was possible to obtain 90% of the initial IgG, IgA and IgM within 85, 119, and 160 min, based on 1 m2 of membrane area and 50 L of skim milk volume. The long-term process exposure at 50 °C did not affect α-La and IgG but β-Lg (3–5% denaturation), which, however, was selectively retained by the MF. In conclusion, MF is not only suitable for fractionation of the major whey proteins and caseins, but also for the minor and far bigger immunoglobulins.

Milk protein fractionation for casein/whey protein separation by microfiltration in diafiltration mode was investigated using washing waters differing in pH, calcium content and ionic strength. Properties of the deposit layer and process efficiency were assessed as functions of diafiltration progress and deposited layer structures were analysed. Results were compared with findings obtained in crossflow mode with a gradual change from milk to diafiltration medium. Deionised water as well as softened water induced a significant increase of deposited casein layer hydration, thereby increasing flux levels and separation efficiency. Dependent on water hardness, tap water was found to induce fewer changes to the deposit hydration and process performance indicating less change of casein micelles. These findings are consistent with the generic colloidal behaviour of casein micelles as previously determined through laboratory experiments. Therefore, waters with a certain degree of hardness could be recommended as the most suitable diafiltration medium not requiring pre-treatment.

Compared to tubular membranes, spiral-wound membranes (SWM) cannot be operated at very high crossflow velocities due to mechanical instabilities of the module. We therefore applied a novel approach using custom-made microfiltration SWM modules to study the effects of crossflow velocities beyond the level currently applicable for commercial SWM. First, we used a modified industrial SWM (0.96 m) to measure flux and protein permeation spatially resolved within the limits of crossflow conditions applied in routine operation (maximum ΔpL of 1.3 bar). Secondly, we applied a short SWM (0.24 m) to assess the effect of extreme crossflow velocities with a corresponding ΔpL of up to 2.8 bar m−1. The goal was to investigate the effects of deposit formation reduced to the minimum, and thus, to measure the theoretically achievable improvement of flux and protein permeation even though this is currently not practicable. As key result, we found a critical crossflow velocity up to which a significant increase of protein mass flow could be achieved; beyond which, however, no further reduction in filtration resistance caused by deposit formation was observed. The filtration became independent of the transmembrane pressure. Based on these findings, more rigid modules could potentially improve the performance of SWM.

During microfiltration, the length-dependent transmembrane pressure (ΔpTM) causes an inhomogeneous filtration performance along the module. Elimination of this length effect was found to increase the overall filtration efficiency of ceramic membranes significantly. Up to the authors’ knowledge, a systematic investigation on the spatial behavior of polymeric spiral-wound membranes (SWM) during microfiltration and the potential of a homogeneous ΔpTM has not been carried out yet. A modified SWM with permeate pockets divided in four hermetically separated sections was used to assess the length-resolved filtration performance during skim milk fractionation. Along the membrane, flux and fouling resistance decreased, whereas protein permeation increased. By adjusting the permeate pressure comparable to the uniform transmembrane pressure mode in ceramic membranes, the length dependency of ΔpTM along the module was reduced. In turn, the filtration performance was found to be more balanced. The overall whey protein mass flow increased as all sections were operating at the same ΔpTM under optimal condition. For the first time, we have proved with experimental data that SWM behave similar to ceramic membranes with regard to the length dependency of the filtration performance. Thus, the concept of a length-independent ΔpTM seems promising to enhance filtration performance of SWM at industrial scale.

Author Summary: A dead-end filtration process of skim milk with ceramic hollow fiber membranes was investigated using in situ Magnetic Resonance Imaging. Special emphasis was put on contrast optimization exploiting water relaxation times being sensitive to casein concentration in the formed deposit layer.

Existing works on the influence of spatial effects on flux and permeation of proteins in microfiltration (MF) have focused on ceramic membranes. There is little information on spiral-wound membranes (SWMs). Since the inner core of a SWM is practically inaccessible by non-destructive techniques, three different prototypes were constructed in this study to optimize suitability for the investigation of spatial effects on filtration performance. To measure the pressure drop, shortened SWMs 0.25, 0.50, and 0.75 times the length of a standard industrial SWM (0.96 m) were designed. Second, a sectioned membrane (0.96 m) with separated compartments on the permeate side was constructed to analyze spatial effects on flux and protein permeation along the flow path of a SWM. Three different features characterized this sectioned module: sectioned permeate pockets, a sectioned permeate collection tube, and sectioned permeate drain and measurement systems. Crossflow filtration experiments showed that these modifications did not alter the filtration performance compared to an unmodified control SWM. Thus, it can be applied to assess spatially-resolved filtration performance in SWMs. The third prototype designed was a test cell with accessible flat sheet membranes and spacer material, as in SWMs. The flow path in this test cell was designed to match the characteristics of the channels between the membrane sheets in a standard SWM as closely as possible. The flow path length and the combination of membrane material and spacer architecture were the same as in the control SWM. This test cell was designed to assess the effects of length and processing conditions on the formation of a deposit layer. The combined results of these test modules can yield new insights into the spatial distribution of flux, permeation of target components, and deposit formation.

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&ndash, 0.8 &micro, m), transmembrane pressures (0.5&ndash, 2.5 bar), and temperatures (10, 50 &deg, C) were investigated. The transmission of immunoglobulin G (IgG) and casein during the filtration of raw skim milk (&lt, 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 &beta, 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 &micro, m 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 &micro, m yielded fluxes up to 35% higher than 0.2 &micro, m 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.

The cancer/testis antigen (CT) family is defined by its specific expression pattern. In most cases CT antigens are expressed in normal human tissue only in germline cells and some tumors. Because of their restricted expression pattern CT antigens are regarded as potential targets for vaccine immunotherapy. Little is known about the functions of the various CT antigens. Some of these CT antigens (CT 7; MAGE A3/6) seem to be involved in the dysregulation of cell-cycle control and increased cell proliferation. The monoclonal antibody Ki-A10 detects a nuclear antigen with a unique distribution pattern in normal human tissues and tumors. The antigen is now characterized as cancer/testis antigen 45 (CT45). The accurate localisation of CT45 could provide details about its function. Therefore immunfluorescence stainings with subsequent confocal laser microscope analysis were performed to deliver precise data about the nuclear localisation of CT45. Different human tumor celllines (L428, HT1080, WS1-CLS, SW872) and 5-aza-2'-deoxycytidine treated human peripheral blood lymphocytes were stained with mab Ki-A10. All stainings showed an irregular and dotlike pattern, which is wellknown for nuclear speckles (interchromatin granule clusters). Nuclear speckles are dynamic structures known to harbour many proteins of the pre-mRNA splicing machinery. In addition CT45 shows a clear homology to a DEAD-Box protein (DDX26). DEAD-Box proteins are generally believed to be RNA helicases, which in turn are localised in nuclear speckles. Due to these results we believe that CT45 could be involved in pre-mRNA splicing.

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.

Ki-Mcm6, a New Monoclonal Antibody Specific to Mcm6: Comparison of the Distribution Profile of Mcm6 and the Ki-67 Antigen

BACKGROUND: Proper treatment of lymph node-negative breast cancer depends on an accurate prognosis. To improve prognostic models for this disease, we evaluated whether an immunohistochemical marker for proliferating cells, Ki-S2 (a monoclonal antibody that binds to a 100-kd nuclear protein expressed in S, G2, and M phases of the cell cycle), is an accurate indicator of prognosis.METHODS: We studied 371 Swedish women with lymph node-negative breast cancer; the median follow-up time was 95 months. The fraction of tumor cells in S phase was assessed by flow cytometry, and tumor cell proliferation was measured immunohistochemically with the monoclonal antibodies Ki-S2 and Ki-S5 (directed against the nuclear antigen Ki-67). A combined prognostic index was calculated on the basis of the S-phase fraction, progesterone receptor content, and tumor size.RESULTS: In multivariate analyses that did or did not (263 and 332 observations, respectively) include the S-phase fraction and the combined prognostic index, the Ki-S2 labeling index (percentage of antibody-stained tumor cell nuclei) emerged as the most statistically significant predictor of overall survival, disease-specific survival, and disease-free survival (all two-sided P

Background Due 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. Methods CT45 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. Results Reduced 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. Conclusions Providing 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.

Cancer/testis (CT) antigens are members of a large tumor-associated protein family of increasing interest because of their diagnostic and possible therapeutic implications. Only few studies have investigated CT antigen expression in Hodgkin’s lymphoma (HL) so far. Recently, immunopurification of a 25/22-kDa antigen and sequencing revealed a peptide of 14 amino acids, which corresponds to the gene product of the newly described gene family MGC27005, located on chromosome Xq26.3, now termed CT45. The antigen shows a CT antigen-like expression pattern in human tissues and is detectable by the monoclonal antibody Ki-A10 generated after immunization of mice with HL-derived cell line L428. In this study, the expression pattern of CT45 in a large number of patients with HL included in subsequent pediatric multicenter treatment protocols of the GPOH was analyzed. Immunohistochemical results of Ki-A10 staining were correlated with histological subtype, immunophenotype, clinical data and outcome. In total, 1124 children and adolescents with HL diagnosed between 1978 and 2004 were studied. Median age of the patients was 13.7 years (range, 2.2 to 19.5) and 637 patients 02(57%) were male. Classical HL (cHL) was diagnosed in 988 patients (88%) and nodular lymphocyte predominant HL (NLPHL) was seen in 136 patients (12%). The group of cHL included 683 cases of nodular sclerosis HL (NSHL), 280 cases of mixed cellularity HL (MCHL), ten cases of lymphocyte-rich cHL (LRCHL), seven cases of lymphocyte-depleted HL (LDHL) and eight cases without subclassification. Nuclear CT45 expression was found in 526 cases (47%) with striking differences among histological subtypes and unrelated to CD30, CD20 and latent EBV infection. In NLPHL, 15% of cases scored CT45 positive in contrast to 51% of cases in cHL (p CT45 expression, which is restricted to neoplastic cells in HL, is found in a significant number of children and adolescents with HL, especially in cases of NSHL, aggressive histological subtypes and advanced disease. Due to effective polychemotherapy with or without low-dose radiotherapy, CT45 status did not emerge as an adverse prognostic factor in children and adolescents with HL in this series.