Alteration of Intestinal Microbiome of Clostridioides difficile-Infected Hamsters during the Treatment with Specific Cow Antibodies.

Clostridioides difficile infection (CDI) often develops after pretreatment with antibiotics, which can lead to damage of the intestinal microbiome. The approach of this study was to use specific polyclonal antibodies isolated from the milk of immunized cows to treat CDI, in contrast to the standard application of nonspecific antibiotics. To gain a deeper understanding of the role of the microbiome in the treatment of CDI with bovine antibodies, stool and intestinal fluid samples of hamsters were collected in large quantities from various treatments (>400 samples). The results show that the regeneration of the microbiome instantly begins with the start of the antibody treatment, in contrast to the Vancomycin-treated group where the diversity decreased significantly during the treatment duration. All antibody-treated hamsters that survived the initial phase also survived the entire study period. The results also show that the regeneration of the microbiome was not an antibody-induced regeneration, but a natural regeneration that occurred because no microbiota-inactivating substances were administered. In conclusion, the treatment with bovine antibodies is a functional therapy for both the acute treatment and the prevention of recurrence in hamsters and could meet the urgent need for CDI treatment alternatives in humans.

Data concerning the fractionation of individual whey proteins and casein micelles by microfiltration with ceramic gradient membranes.

Data are related to the research article "Fractionation of casein micelles and immunoglobulins by microfiltration in diafiltration mode Study of the transmission and yield of IgG, IgA and IgM" [1]. The data show the transmission and yield of the individual whey proteins α-Lactalbumin (α-La), ß -Lactoglobulin (ß -Lg), blood serum albumin (BSA), lactoferrin (LF), lactoperoxidase (LPO) and the immunoglobulins IgG, IgA, IgM during microfiltration (0.14 µm) performed in diafiltration mode at 50 °C with different applied transmembrane pressures (0.6-3 bar). The data provide information on the decrease of the respective proteins in the microfiltration retentate and their increase in the UF retentate. The relevant analytical methods for the individual protein detection were performed by reversed phase high performance liquid chromatography and ELISA. The isoelectric point of IgG and IgM was measured with the Zetasizer Nano ZS.

Technical Concepts for the Investigation of Spatial Effects in Spiral-Wound Microfiltration Membranes.

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.

Milk Protein Fractionation by Means of Spiral-Wound Microfiltration Membranes: Effect of the Pressure Adjustment Mode and Temperature on Flux and Protein Permeation.

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.

Treatment and Prevention of Recurrent Clostridium difficile Infection with Functionalized Bovine Antibody-Enriched Whey in a Hamster Primary Infection Model.

Toxin-induced Clostridium difficile infection (CDI) is a major disease characterized by severe diarrhea and high morbidity rates. The aim with this study was to develop an alternative drug for the treatment of CDI. Cows were repeatedly immunized to establish specific immunoglobulin G and A titers against toxins A (TcdA) and B (TcdB) and against C. difficile cells in mature milk or colostrum. The effect of three different concentrations of anti-C. difficile whey protein isolates (anti-CD-WPI) and the standard of care antibiotic vancomycin were investigated in an animal model of CD infected hamsters (6 groups, with 10 hamsters each). WPI obtained from the milk of exactly the same cows pre-immunization and a vehicle group served as negative controls. The survival of hamsters receiving anti-CD-WPI was 50, 80 and 100% compared to 10 and 0% for the control groups, respectively. Vancomycin suppressed the growth of C. difficile and thus protected the hamsters at the time of administration, but 90% of these hamsters nevertheless died shortly after discontinuation of treatment. In contrast, the surviving hamsters of the anti-CD-WPI groups survived the entire study period, although they were treated for only 75 h. The specific antibodies not only inactivated the toxins for initial suppression of CDI, but also provoked the inhibition of C. difficile growth after discontinuation, thus preventing recurrence. Oral administration of anti-CD-WPI is a functional therapy of CDI in infected hamsters for both primary treatment and prevention of recurrence. Thus, anti-CD-WPI could address the urgent unmet medical need for treating and preventing recurrent CDI in humans.

Data concerning the chromatographic isolation of bovine IgG from milk- and colostral whey.

Data included are related to the research article "Isolation of biofunctional bovine immunoglobulin G from milk- and colostral whey with mixed-mode chromatography at lab and pilot scale" (Heidebrecht et al., 2018) [1]. Data show individual bovine whey proteins in flow-through and elution fractions using different chromatographic resins as well as different binding and elution conditions. The relevant analytical methods for individual protein detection were SDS-PAGE and reversed phase- high performance liquid chromatography. The focus of the data is on the two mixed mode materials MEP HyperCel™ and Capto™-multimodal chromatography. Resins were used individually, in series and at different scale. Data provide information at which binding and elution conditions it is possible to isolate bovine IgG from milk and colostral whey and at which purity.

Concentration of Immunoglobulins in Microfiltration Permeates of Skim Milk: Impact of Transmembrane Pressure and Temperature on the IgG Transmission Using Different Ceramic Membrane Types and Pore Sizes.

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.

Isolation of biofunctional bovine immunoglobulin G from milk- and colostral whey with mixed-mode chromatography at lab and pilot scale.

The aim of the present work was to develop a new scalable and cost-efficient process to isolate bovine immunoglobulin G from colostral whey with high purity and minimal loss of activity. The mixed mode material Mercapto-Ethyl-Pyridine-Hypercel™ was identified appropriate for direct capture of immunoglobulin G. The binding mechanism is primarily based on hydrophobic interactions at physiological conditions. As compared to immunoglobulin G, all other low molecular whey proteins such as α-Lactalbumin or ß-Lactoglobulin, except lactoperoxidase, are more hydrophilic and were therefore found in the flow-through fraction. In order to remove lactoperoxidase as an impurity the column was combined in series with a second mixed mode material (Capto™- with N-benzoyl-homocysteine as ligand) using the same binding conditions. At pH 7.5 the carboxyl group of this ligand is negatively charged and can hence bind the positively charged lactoperoxidase, whose isoelectric point is at pH 9.6. After sample application, the columns were eluted separately. By combining the two columns it was possible to obtain immunoglobulin G with a purity of >96.1% and yield of 65-80%. The process development was carried out using 1 mL columns and upscaling was performed in three steps up to a column volume of 8800 mL for the Hypercel™ column and 3000 mL for the Capto™- column. At this scale it is possible to obtain 130-150 g pure immunoglobulin G from 3 L colostrum within five hours, including the regeneration of both columns. Additionally, the impact of freeze-drying on the isolated immunoglobulin G was studied. The nativity of the freeze dried immunoglobulin was above 95%, which was proven by reversed phase liquid chromatography and validated by differential scanning calorimetry. The activity of immunoglobulin G was preserved over the isolation process and during drying as measured by enzyme-linked immunosorbent assay. In conclusion, by applying the proposed isolation process, it becomes feasible to obtain pure, active and stable imunnunoglobulin G at large scale.

Lysyl oxidase like-4 monoclonal antibody demonstrates therapeutic effect against head and neck squamous cell carcinoma cells and xenografts.

A new member of the lysyl oxidase (LOX) family, lysyl oxidase-like 4 (LOXL4), is overexpressed in head and neck squamous cell carcinoma (HNSCC) compared to normal squamous epithelium. A monoclonal antibody (mAb) derived from fusion of Balb/c mouse splenocytes immunized with LOXL4 specific peptide was used to evaluate its therapeutic efficacy in 15 HNSCC cell lines associated with LOXL4 overexpression. For xenograft experiments 41 severe combined immunodeficient (SCID) mice were used to analyze LOXL4-mAb mediated tumor regression. Cell viability was analyzed using cytotoxicity-, and clonogenic-assays. Significant suppression of tumor cell growth was observed in 12 out of 15 (80%) tumor cell lines after 48 hr exposure to the mAb (LD50 of 15 µg/ml to 45 µg/ml). The effect induced by the antibody could be blocked by pre-incubation of the antibody with the peptide used for immunization of the mice and antibody generation, indicating that the effect of the antibody is specific. In mice inoculated with HNSCC cells, i.v. injections of the LOXL4-mAb resulted within 70 days in extensive tumor destruction in all treated animals whereas no tumor regression occurred in control animals. In mice pre-immunized i.v. with LOXL4-mAb and subsequently injected with HNSCC cells, tumor development was considerably delayed in contrast to non LOXL4-mAb pre-immunized animals. These results demonstrate that the LOXL4-mAb has potent antitumor activity and suggest its suitability as a therapeutic immune agent applicable to HNSCC exhibiting tumor specific upregulation of LOXL4.

Down-regulation of the cancer/testis antigen 45 (CT45) is associated with altered tumor cell morphology, adhesion and migration.

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.

Regulation of repp86 stability by human Siah2.

Human repp86 is a nuclear protein that is expressed in a tightly limited period of time during the cell cycle and plays an essential role in its progression. Manipulation of repp86 expression by reduction of endogenous repp86 or overexpression of exogenous repp86 results in cell cycle arrest. We found that repp86 interacts with human Siah2, which is a known mediator for proteasomal degradation. Siah2 failed to interact with repp86 lacking the first 67 N-terminal amino acids. Overexpression of Siah2 reduced endogenous and exogenous repp86 at the protein level without affecting its mRNA, as shown by cotransfection and RT-PCR experiments. Furthermore, MG-132--a specific inhibitor of the proteasome--blocked the degradation of repp86 in Siah2 overexpressing cells. Moreover, transiently transfected Siah2 abrogated the mitotic arrest in repp86 overexpressing cells. Our data show that Siah2 is an important mediator of repp86 protein degradation.

Human EML4, a novel member of the EMAP family, is essential for microtubule formation.

Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1-249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [3H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.

Characterization and expression of CT45 in Hodgkin's lymphoma.

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.

Repp86 expression and outcome in patients with neuroblastoma.

PURPOSE: Given the well-known challenges of neuroblastoma prognosis, we investigated whether the expression of restrictedly expressed proliferation-associated protein of 86 kDa theoretical molecular mass (repp86), a proliferation-associated protein expressed in S, G2, and M phases of the cell cycle, correlates with the clinical outcome in patients with neuroblastoma. PATIENTS AND METHODS: 161 children with different stages of neuroblastoma were studied; the median follow-up time was 72.8 months. The patients were staged according to the International Neuroblastoma Staging System, and histologic grading of the tumors was performed according to the criteria of Hughes and those of the International Neuroblastoma Pathology Classification. The MYCN gene copy number was determined by Southern blot analysis or fluorescence in situ-hybridization, and repp86 expression was assessed immunohistochemically by means of monoclonal antibody Ki-S2 on paraffin sections from archival tumor samples. RESULTS: A repp86 labeling index (RI) of more than 10% positive tumor cells significantly predicted a shortened disease-free interval and an increased tumor mortality (both P <.0001). Moreover, the RI allowed the identification of patients with favorable and adverse prognosis in subsets defined by stage, grade, age, and MYCN status. In a multivariate analysis, the RI emerged as the most important predictor of event-free and disease-specific survival with hazard ratios of 11.7 and 10.5, respectively (both P <.0001). CONCLUSION: It seems that repp86 expression is closely associated with the biologic behavior of neuroblastoma. Assessment of the RI might, therefore, considerably refine prognostic models.

repp86: A human protein associated in the progression of mitosis.

Human repp86 becomes detectable in the nucleoplasm of cycling cells at the G(1)-S boundary, condenses at the centrosomes with the onset of mitosis, during which it progressively locates to the mitotic spindle and to the midbody, and vanishes at the completion of cytokinesis. The repp86 cDNA was cloned and sequenced. Full-length repp86 and its COOH-terminal domain cosediment with polymerized microtubules, linking repp86 to the family of microtubule-associated proteins. During prophase and metaphase, repp86 interacts on the mitotic spindle with the putative motor protein Hklp2. Thus, repp86 may function in targeting Hklp2 to the microtubule minus ends, its activity being regulated by phosphorylation of serine/threonine residues. Exogenous overexpression of repp86 provokes accumulation of cells in G(2)-M phase and subsequent polyploidization, suggesting that excess repp86 may interfere with correct nuclear division.