Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration

Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.     

Podophyllum hexandrum modulates gamma radiation-induced immunosuppression in Balb/c mice: Implications in radioprotection

Aqueous extract of Podophyllum hexandrum (RP-1), which has been reported to render more than 82% survival against whole body lethal (10 Gy) gamma-irradiation in mice, was further investigated for its immunomodulatory potential. In this study, no significant change could be scored in peritoneal macrophages survival up to 8th day after whole body irradiation. RP-1 treatment (200 mg/kg body weight, i.p.) alone or 2 h before whole body irradiation enhanced macrophage survival significantly (p < 0.05) as compared to irradiated control mice. In irradiated animals, there was significant (p < 0.01) reduction in splenocyte survival and proliferation as revealed by 3H-TdR method. RP-1 treatment (200 mg/kg) alone or 2 h before irradiation countered the decrease in survival of splenocytes and proliferation significantly (p < 0.05) as compared to irradiated control group. Whole body irradiation also significantly (p < 0.05) reduced the population of CD4+ and CD8+ T cells and bone marrow GM-CFU at 24 h and 72 h post-irradiation intervals, respectively, as compared to unirradiated control. RP-1 treatment 2 h before whole body irradiation countered the decrease in CD4+ and CD8+ T cells populations and CGM-CFU. Nitric oxide free radicals generation was enhanced significantly (p < 0.05) in the supernatant of peritoneal macrophage cultures exposed to 2 Gy gamma radiation ex vivo in comparison to unirradiated control, which was reduced by pre-irradiation (−2 h) administration of RP-1. Whole body irradiation (10 Gy) also reduced the serum titres of IL-3, IL-1 and various IgG isotypes observed at different post-irradiation time interval. RP-1 treatment alone or before whole body irradiation countered radiation induced decrease in the titre of IL-1, IL-3 and IgG’s in the serum of mice. These findings indicate immunostimulatory potential of RP-1.     

Review: passive immunity in beef-suckler calves

Colostrum-derived passive immunity is central to the health, performance and welfare of neonatal beef-suckler calves, and economics of beef-farming enterprises. Compared to dairy calves, mainly Holstein-Friesian, there is much less research carried out on passive immunity and associated factors in beef calves. Thus, this review aimed to summarise and interpret published information and highlight areas requiring further research. The transfer of immunoglobulin G1 (IgG1) from blood to mammary secretions is greater for beef × dairy cows compared to most beef breed types. Considerable between-animal variance is evident in first-milking colostrum yield and immunoglobulin concentration of beef-suckler cow breed types. First-milking colostrum immunoglobulin concentrations are similar for within-quarter fractions and for the front and rear quarters of the udder. First-milking colostrum yield is higher for beef × dairy cows than beef × beef and purebred beef breeds, and higher for multiparous than primiparous cows, but generally colostrum immunoglobulin concentration is relatively similar for each of the respective categories. Consequently, colostrum immunoglobulin mass (volume × concentration) production in beef cows seems to be primarily limited by colostrum volume. The effect of maternal nutrition during late gestation on colostrum yield is not well documented; however, most studies provide evidence that colostrum immunoglobulin concentration is not adversely affected by under-nutrition. Factors that impinge upon the duration between birth and first suckling, including dam parity, udder and teat anatomy and especially dystocia, negatively impact on calf passive immunity. Colostrum immunoglobulin mass ingested relative to birth weight post-parturition is the most important variable determining calf passive immunity. Research indicates that feeding the beef calf a colostrum volume equivalent to 5% of birth weight shortly after parturition, with subsequent suckling of the dam (or a second feed) 6 to 8 h later, ensures adequate passive immunity, equivalent to a well-managed suckling situation. Within beef-suckler cow genotypes, calf passive immunity is similar for many common beef breeds, but is generally higher for calves from beef × dairy cows. Compared to older cows, calves from younger cows, especially primiparous animals, have lower serum immunoglobulin concentrations. Most studies have shown no adverse impact of maternal dietary restriction on calf passive immunity. The prevalence of failure of passive transfer (FPT) in beef calves varies considerably across studies depending on the test used, and what cut-off value is assumed or how it is classified. The accuracy and precision of methodologies used to determine immunoglobulin concentrations is concerning; caution is required in interpreting laboratory results regarding defining colostrum ‘quality’ and calf passive immune ‘status’. Further research is warranted on colostrum-related factors limiting passive immunity of beef calves, and on the validation of laboratory test cut-off points for determining FPT, based on their relationships with key health and performance measures.     

Experimental trichinosis-II. Quantitative and qualitative variations of serum in CBA mouse and Wistar rat (author's transl)]

Variations of serum globuline and immunoglobulins were measured by electrophoresis on cellulose acétate and radial immunodiffusion during the course of infection in inbred CBA mice and Wistar rats.In the two animal species, electrophoresis showed a significant decrease of serum albumin attending an increase of γ globulins. This phenomenon was earlier and greater in mice than in rats. This difference might be explained by suggesting that Trichinella is better adapted to rats than to mice.Beginning in the second week of infection, all classes of immunoglobulins were increased in the mouse with IgG_2a and IgA showing the greatest increase. The different classes of immunoglobulins continued to increase during the 4th month of infection but the rate of increase diminished after the second month except for IgA which increased between the 60th and 120th day.     

Effects of 2.5‐hour sumo training on serum opsonic activity

Sumo is a traditional Japanese sport, but the effect of actual daily training on neutrophil function is unknown. We evaluated the effect of sumo training on serum opsonic activity (SOA), which is one of the main neutrophil‐related functions. Seventeen male university sumo wrestlers participated in the study. Changes in anthropometric parameters, concentrations of serum immunoglobulins (IgG, IgA and IgM), complements (C3 and C4), myogenic enzymes (lactate dehydrogenase, asparate aminotransferase, alanine aminotransferase and creatine kinase), white blood cell/neutrophil counts and SOA were measured immediately before and after actual daily training for 2.5 h. Compared with the pre‐values, immunoglobulins and complements, myogenic enzymes and white blood cell/neutrophil counts significantly increased (p <0.01 for all). As for SOA, the values of the peak height and the area under the curve significantly increased after the training when assessed using lucigenin as a chemiluminigenic probe (p <0.01 for all), but showed no significant change when luminol was used as the chemiluminigenic probe. In conclusion, daily actual sumo training for 2.5 h increases SOA, thus possibly activating the reactive oxygen species production of neutrophils. Copyright © 2009 John Wiley & Sons, Ltd.     

Angiogenic transforming capacity of IgG purified from plasma of type 1 diabetic patients

We previously demonstrated that plasma of type 1 diabetic patients contains antibodies complexed irreversibly with Grp94 that also display proteolytic activity. In this work, we wanted to test whether antibodies obtained from diabetic plasma may convey an inflammatory risk on vascular cells. To this aim, IgG were purified on the Protein-G column from individual plasma of eight type 1 diabetic patients, and then tested on HUVECs to measure effects on cell growth and morphologic changes at different incubation times. The purified fractions of IgG contained a significant amount of Fab/(Fab)₂, both free and in big aggregates, and anti-Grp94 antibodies, mostly irreversibly linked with, but also free of Grp94. The purified fractions of both Fab/(Fab)₂ and whole IgG stimulated the proliferation and sustained the angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) at sub-nanomolar concentrations. IgG from normal plasma neither stimulated the cell growth nor induced any differentiation of HUVECs. The maximum cell growth stimulation occurred at 6–9 hrs and associated with the strong activation of the ERK1/2 pathway, whereas angiogenic transformation was completed later when the ERK1/2 activation was silenced and cell growth stimulation significantly reduced. Neither proteolytic activity of MMP-9 nor VEGF were apparently involved in mediating the angiogenic differentiation of HUVECs that mostly correlated with an increased expression of HSP70 closely coupled with cell membrane-bound inactive species of MMP-9. Results indicate that effects displayed on HUVECs by antibodies purified from diabetic plasma are likely sustained by immune complexes with Grp94 that may thus predict an increased risk of angiogenic transformation in vivo .