Procedure for concentration and conditioning of hybridoma cell culture medium for monoclonal antibody purification

Summary Concentration and conditioning the hybridoma cell culture medium is an important step in the monoclonal antibody purification procedure. This report describes a frequent first-step concentration of the hybridoma cell culture medium and the conditioning of the concentrated medium for the affinity purification of the monoclonal antibodies.     

The enzymatic degradation of polymers in vitro

Specimens of 14C-labeled poly(ethylene terephthalate), nylon 66, and poly(methyl methacrylate) have been synthesized and exposed, in vitro, to a number of enzyme solutions. Poly(ethylene terephthalate) was found to be affected by esterase and papain, although in different ways, but not by trypsin or chymotrypsin. Nylon 66 was unaffected by esterase but degraded by the other three. Poly(methyl methacrylate) was not affected by any of these enzymes. This indicates that some nominally stable polymers are susceptible to degradation by enzymes under some circumstances. The amount of degradation is small, but could have significant sequelae should it be reproduced in vivo.     

Crossing the barrier: Evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis and S. oralis

A paradigm for Streptococcus interspecies gene transfer is represented by the mosaic pbp genes encoding the target enzymes for beta-lactam antibiotics, the penicillin-binding proteins, in Streptococcus pneumoniae. We investigated a collection of oral streptococci from three continents by comprehensive multi-locus sequence typing analysis in order to trace the origin of a mosaic block belonging to a dominant family of mosaic pbp2x implicated in penicillin resistance of S. pneumoniae. One widespread family of mosaic pbp2x occurred in all three distinct clusters of S. pneumoniae, Streptococcus mitis and Streptococcus oralis, documenting independent inter- and intraspecies recombination events. Moreover, potential ancestor genes of this mosaic block could be identified in two penicillin-susceptible S. mitis strains from South Africa and Spain, facilitating the identification of pbp2x mutations relevant for resistance development.     

Protection of granulocytes by vitamin E in glutathione synthetase deficiency.

The administration of vitamin E (alpha-tocopherol) was found to improve polymorphonuclear leukocyte function in an infant with congenital deficiency of glutathione synthetase activity. Before therapy with vitamin E the abnormal leukocytes exposed to phagocytic challenge showed oxidant damage. They released 60 per cent more hydrogen peroxide than did normal leukocytes, iodinated only 20 to 25 per cent of the normal number of particles, and were unable to kill bacteria as effectively as normal leukocytes although the rates of phagocytosis were normal. These functional abnormalities disappeared when the patient was placed on 400 IU of alpha-tocopherol daily for three months. Associated with the functional improvement was a normalization of microtubule assembly during phagocytic challenge.     

Ontogenesis of leptin expression in different adipose tissue depots in the rat

Serum leptin levels and leptin mRNA expression by adipose tissue increase with age and are mainly associated with an increase in adiposity. Regional changes in both leptin production and fat distribution contribute to circulating leptin levels and may play a role in the regulation of body weight. a capacity that changes during development. Here, we have studied leptin mRNA expression in four different white adipose tissue depots (epididymal, retroperitoneal, mesenteric, inguinal; namely, EWAT, RWAT, MWAT, IWAT) and in interscapular brown adipose tissue (IBAT). We have also studied their relationship with lipid content and adiposity changes, together with serum leptin levels in male rats at different ages (18, 55, 93, 159, 212, 294 and 355 days). Serum leptin levels increased during development, reaching stable levels at the age of 7 months, and, as expected, were highly correlated with both the adiposity index (r=0.908, P<0.01) and body weight (r=0.906, P<0.01). Leptin mRNA expression also increased with age, following characteristic ontogenic patterns in every adipose tissue depot. The patterns were similar in EWAT and RWAT: leptin expression increased very rapidly during the first 55 days for EWAT and 3 months for RWAT, with a peak in the latter at 7 months, and high expression levels were retained for the rest of the study period. In IWAT and IBAT, leptin expression increased steadily during the 12-month period studied and was significantly lower than levels in EWAT and RWAT. Leptin expression in MWAT increased progressively with age to reach levels close to those of EWAT and RWAT in 10-month-old animals. The pattern of leptin expression in both EWAT and RWAT paralleled their lipid content, and leptin mRNA expression per unit of tissue lipid content was maintained high and constant from a very young age (about 2 and 3 months, respectively). However, the expression of mRNA for leptin (expressed per unit of tissue lipid concentration) in MWAT, IWAT and IBAT increased steadily during the whole period studied, without attaining the maximal levels observed in EWAT and RWAT. MWAT, IWAT and IBAT maintained their capacity to increase leptin mRNA expression in response to an additional accumulation of lipids. Our data demonstrate that there are regional-specific differences and different rates of increase of leptin gene expression within distinct depots of WAT and BAT. These changes cannot be uniquely explained by changes in adiposity or lipid content, implying that there are regional-specific regulatory mechanisms that may depend on the attenuation with age of the beta-adrenergic inhibitory signalling pathway upon leptin expression or on other factors.     

Brain reactivity of lymphocytotoxic antibodies in systemic lupus erythematosus with and without cerebral involvement.

A prospective clinical study of cold-reactive lymphocytotoxic antibodies in systemic lupus erythematosus has been completed. A highly significant association between serum lymphocytotoxicity and the development of cerebral manifestations was observed. While lymphocytotoxic antibodies from patients with cerebral lupus were absorbed by homogenates of human brain, those from patients who at no time had evidence of cerebral disease failed to cross-react with brain. It is suggested that subpopulations of lymphocytotoxic antibodies differ in their brain reactivity, and that one population may be causally related to the development of some of the features of cerebral lupus.     

Long-term culture of human bone marrow stromal cells in the presence of basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells. Normally, large numbers of human bone marrow stromal cells are difficult to obtain. However, nanogram/ml concentrations of bFGF stimulate the growth of passaged bone marrow stromal cells both in media formulated for optimal growth of stromal cells and in a simple mixture of RPMI-1640 and 10% fetal calf serum facilitating the successive expansion of stromal cells through multiple passages. bFGF also greatly accelerates the formation of a primary stromal cell layer following inoculation of newly harvested bone marrow cells into dishes. In the presence of bFGF, the stromal cells attain high densities, lose their contact inhibition and grow in multilayered sheets. Heparin greatly potentiates the stimulatory effect of low concentrations of bFGF. The effects of bFGF are fully reversible: cells cultured in the presence of this factor for multiple passages revert to normal growth rates following trypsinization and subculture. A short (4 h) exposure of the cells to bFGF elicits profound growth stimulation. This supports the hypothesis that this factor binds to glycosaminoglycans in the cell matrix which act as a storage reservoir for this cytokine.